Alexander R. Moise

ISX is a retinoic acid-sensitive gatekeeper that controls intestinal β,β-carotene absorption and vitamin A production

The uptake of dietary lipids from the small intestine is a complex process that depends on the activities of specific membrane receptors with yet unknown regulatory mechanisms. Using both mouse models and human cell lines, we show here that intestinal lipid absorption by the scavenger receptor class B type 1 (SR‐BI) is subject to control by retinoid signaling. Retinoic acid via retinoic acid receptors induced expression of the intestinal transcription factor ISX. ISX then repressed the expression of SR‐B1 and the carotenoid‐15,15′‐oxygenase Bcmo1. BCMO1 acts downstream of SR‐BI and converts absorbed β,β‐carotene to the retinoic acid precursor, retinaldehyde. Using BCMO1‐knockout mice, we demonstrated increased intestinal SR‐BI expression and systemic β,β‐carotene accumulation. SR‐BI‐dependent accumulation of β,β‐carotene was prevented by dietary retinoids that induced ISX expression. Thus, our study revealed a diet‐responsive regulatory network that controls β,β‐carotene absorption and vitamin A production by negative feedback regulation. The role of SR‐BI in the intestinal absorption of other dietary lipids, including cholesterol, fatty acids, and tocopherols, implicates retinoid signaling in the regulation of lipid absorption more generally and has clinical implications for diseases associated with dyslipidemia.—Lobo, G. P., Hessel, S., Eichinger, A., Noy, N., Moise, A. R., Wyss, A., Palczewski, K., von Lintig, J. ISX is a retinoic acid‐sensitive gatekeeper that controls intestinal β,β‐carotene absorption and vitamin A production. FASEB J. 24, 1656–1666 (2010). www.fasebj.org

Mechanistic aspects of carotenoid biosynthesis.

Carotenoids represent a large class of terpenoids characterized by an extensively conjugated polyene chain. The conjugation system imparts carotenoids with excellent light absorbing properties in the blue-green (450–550 nm) range of the visible spectrum. The light-absorbing properties of carotenoids have been exploited by photosynthetic organisms to extend the range of light absorption by the photosynthetic apparatus beyond that of chlorophyll. 1 Following light absorption the carotenoid excited state undergoes excitation energy transfer to chlorophyll. 2 In addition to their role as accessory pigments, carotenoids protect against excess light by quenching both singlet and triplet state chlorophylls. In plants oxygenated carotenoids, known as xanthophylls, provide additional photoprotective functions by non-photochemical quenching (NPQ) of chlorophyll fluorescence. 2a,3 Carotenoids carry out light independent functions in scavenging peroxyl radicals and preventing oxidative damage particularly against singlet oxygen (1O2).4 Since many organisms and specific animal tissues, such as the macula lutea and the corpus luteum, accumulate large amounts of carotenoids, it was proposed that carotenoids may protect against the damaging effects of oxidative stress in such tissues. 5a,b These properties have prompted much research in the chemopreventative potential of carotenoids. At high oxygen pressures, however, some carotenoids display prooxidative activity4b and some β-carotene formulations have even shown adverse effects in supplementation trials aimed at preventing lung cancer in smokers. 6 The length of the carotenoid polyene chain corresponds to the width of the phospholipid bilayer, which led to the proposal that carotenoids act as “molecular rivets” to stabilize and add rigidity to the phospholipid membrane. 2b,7 The membrane spanning topology could also allow carotenoids to counteract oxidative damage on either side of the membrane. 4b It has also been proposed that membrane-spanning carotenoids can mediate proton transfer across the membrane or serve as transmembrane radical channels. 8 Due to their striking and rich color carotenoids are important floral pigments serving to attract pollinators and seed dispersers. 9 In birds and fish, carotenoids are an important signal of good nutritional condition and are used in ornamental displays as a sign of fitness and to increase sexual attractiveness. 10a,b,c,10d–f Following oxidative cleavage, carotenoids generate apocarotenoid metabolites which serve important signaling and photoreceptive functions. The 11-cis isomers, 11-cis-retinaldehyde, 3,4-didehydro-11-cis-retinaldehyde or 3-hydroxy-11-cis-retinal are used by most animals as a photosensitive moiety coupled to the opsin protein, rhodopsin, cone opsin or melanopsin. 11 These photoreceptor molecules mediate vision and circadian photoentrainment. 12 Bacteria use the light-sensitive carotenoid cleavage product, retinaldehyde, coupled to bacteriorhodopsin and related proteins to transport protons and other ions across the membrane. This ion transport function allows the cell to generate energy, regulate ion balance or sense light. 13a,b,c The acidic forms of several apocarotenoids act as signaling molecules in fungi, plants and vertebrates. The apocarotenoid, trisporic acid, signals mating type in fungi. 14 Plants cleave carotenoids such as 9-cis-neoxanthin to generate the hormone abscisic acid, which plays important roles in inducing seed dormancy, and allowing the plant to adapt to abiotic stress. 15a,b,c Other plant apocarotenoid metabolites, such as strigolactones trigger seed germination of parasitic weeds and inhibit shoot branching. 16 Finally, vertebrates use retinoic acid, a ligand for nuclear receptors to regulate gene transcription in physiological processes that include embryonic development, cell differentiation, and immunity. 17a–c,17d,e Carotenogenesis occurs in all photosynthetic organisms and in some non-photosynthetic bacteria, archaea, protozoa and fungi. Reflecting their ubiquitous presence and pleiotropic roles there are well over 700 different types of carotenoids generated through variations of their pathways of synthesis. 18 There is even recent evidence for the acquisition of carotenogenic enzymes by metazoans through lateral gene transfer from endosymbiotic fungi. 19 Many excellent reviews have focused on the later steps of the carotenoid synthetic pathways and their regulation in bacteria, and plants. 20,21 In this review we concentrate on the mechanisms of carotenoid synthesis by examining the structure and enzymology of enzymes involved in the production of carotenoids starting from the production of isoprenoid precursors.

Retinoid cycle in the vertebrate retina: experimental approaches and mechanisms of isomerization

Retinoid cycle describes a set of chemical transformations that occur in the photoreceptor and retinal pigment epithelial cells. The hydrophobic and labile nature of the retinoid substrates and the two-cell chromophore utilization-regeneration system imposes significant constraints on the experimental biochemical approaches employed to understand this process. A brief description of the recent developments in the investigation of the retinoid cycle is the current topic, which includes a review of novel results and techniques pertaining to the retinoid cycle. The chemistry of the all-trans-retinol to 11-cis-retinol isomerization is also discussed.

Identification of All-trans-Retinol:All-trans-13,14-dihydroretinol Saturase

Retinoids carry out essential functions in vertebrate development and vision. Many of the retinoid processing enzymes remain to be identified at the molecular level. To expand the knowledge of retinoid biochemistry in vertebrates, we studied the enzymes involved in plant metabolism of carotenoids, a related group of compounds. We identified a family of vertebrate enzymes that share significant similarity and a putative phytoene desaturase domain with a recently described plant carotenoid isomerase (CRTISO), which isomerizes prolycopene to all-trans-lycopene. Comparison of heterologously expressed mouse and plant enzymes indicates that unlike plant CRTISO, the CRTISO-related mouse enzyme is inactive toward prolycopene. Instead, the CRTISO-related mouse enzyme is a retinol saturase carrying out the saturation of the 13–14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol. The product of mouse retinol saturase (RetSat) has a shifted UV absorbance maximum, λmax = 290 nm, compared with the parent compound, all-trans-retinol (λmax = 325 nm), and its MS analysis (m/z = 288) indicates saturation of a double bond. The product was further identified as all-trans-13,14-dihydroretinol, since its characteristics were identical to those of a synthetic standard. Mouse RetSat is membrane-associated and expressed in many tissues, with the highest levels in liver, kidney, and intestine. All-trans-13,14-dihydroretinol was also detected in several tissues of animals maintained on a normal diet. Thus, saturation of all-trans-retinol to all-trans-13,14-dihydroretinol by RetSat produces a new metabolite of yet unknown biological function.

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Fatty acid retinyl esters are the storage form of vitamin A (all-trans-retinol) and serve as metabolic intermediates in the formation of the visual chromophore 11-cis-retinal. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, acts by transferring an acyl group from the sn-1 position of phosphatidylcholine to retinol. To define the membrane association and localization of LRAT, we produced an LRAT-specific monoclonal antibody, which we used to study enzyme partition under different experimental conditions. Furthermore, we examined the membrane topology of LRAT through an N-linked glycosylation scanning approach and protease protection assays. We show that LRAT is localized to the membrane of the endoplasmic reticulum (ER) and assumes a single membrane-spanning topology with an N-terminal cytoplasmic/C-terminal luminal orientation. In eukaryotic cells, the C-terminal transmembrane domain is essential for the activity and ER membrane targeting of LRAT. In contrast, the N-terminal hydrophobic region is not required for ER membrane targeting or enzymatic activity, and its amino acid sequence is not conserved in other species examined. We present experimental evidence of the topology and subcellular localization of LRAT, a critical enzyme in vitamin A metabolism.

Identification of a Novel Route of Iron Transcytosis across the Mammalian Blood-Brain Barrier

Objective: This study was undertaken to assess the role of p97 (also known as melanotransferrin) in the transfer of iron into the brain, because the passage of most large molecules is limited by the presence of the blood‐brain barrier, including that of the serum iron transporter transferrin.

Metabolism and transactivation activity of 13,14-dihydroretinoic acid.

The metabolism of vitamin A is a highly regulated process that generates essential mediators involved in the development, cellular differentiation, immunity, and vision of vertebrates. Retinol saturase converts all-trans-retinol to all-trans-13,14-dihydroretinol (Moise, A. R., Kuksa, V., Imanishi, Y., and Palczewski, K. (2004) J. Biol. Chem. 279, 50230–50242). Here we demonstrate that the enzymes involved in oxidation of retinol to retinoic acid and then to oxidized retinoic acid metabolites are also involved in the synthesis and oxidation of all-trans-13,14-dihydroretinoic acid. All-trans-13,14-dihydroretinoic acid can activate retinoic acid receptor/retinoid X receptor heterodimers but not retinoid X receptor homodimers in reporter cell assays. All-trans-13,14-dihydroretinoic acid was detected in vivo in Lrat-/- mice supplemented with retinyl palmitate. Thus, all-trans-13,14-dihydroretinoic acid is a naturally occurring retinoid and a potential ligand for nuclear receptors. This new metabolite can also be an intermediate in a retinol degradation pathway or it can serve as a precursor for the synthesis of bioactive 13,14-dihydroretinoid metabolites.

The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development

Oxidation of retinol via retinaldehyde results in the formation of the essential morphogen all‐trans‐retinoic acid (ATRA). Previous studies have identified critical roles in the regulation of embryonic ATRA levels for retinol, retinaldehyde, and ATRA‐oxidizing enzymes; however, the contribution of retinaldehyde reductases to ATRA metabolism is not completely understood. Herein, we investigate the role of the retinaldehyde reductase Dhrs3 in embryonic retinoid metabolism using a Dhrs3‐deficient mouse. Lack of DHRS3 leads to a 40% increase in the levels of ATRA and a 60% and 55% decrease in the levels of retinol and retinyl esters, respectively, in Dhrs3–/– embryos compared to wild‐type littermates. Furthermore, accumulation of excess ATRA is accompanied by a compensatory 30–50% reduction in the expression of ATRA synthetic genes and a 120% increase in the expression of the ATRA catabolic enzyme Cyp26a1 in Dhrs3–/– embryos vs. controls. Excess ATRA also leads to alterations (40–80%) in the expression of several developmentally important ATRA target genes. Consequently, Dhrs3–/– embryos die late in gestation and display defects in cardiac outflow tract formation, atrial and ventricular septation, skeletal development, and palatogenesis. These data demonstrate that the reduction of retinaldehyde by DHRS3 is critical for preventing formation of excess ATRA during embryonic development.—Billings, S. E., Pierzchalski, K., Butler Tjaden, N. E., Pang, X.‐Y., Trainor, P. A., Kane, M. A., Moise, A. R., The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development. FASEB J. 27, 4877–4889 (2013). www.fasebj.org

Increased adiposity in the retinol saturase-knockout mouse

The enzyme retinol saturase (RetSat) catalyzes the saturation of all‐irans‐retinol to produce (R)‐all‐trans‐13,14‐dihydroretinol. As a peroxisome pro‐liferator‐activated receptor (PPAR) γ target, RetSat was shown to be required for adipocyte differentiation in the 3T3‐L1 cell culture model. To understand the mechanism involved in this putative proadipogenic effect of RetSat, we studied the consequences of ablating RetSat expression on retinoid metabolism and adipose tissue differentiation in RetSat‐null mice. Here, we report that RetSat‐null mice have normal levels of retinol and retinyl palmitate in liver, serum, and adipose tissue, but, in contrast to wild‐type mice, are deficient in the production of all‐trans‐13,14‐dihydroretinol from dietary vitamin A. Despite accumulating more fat, RetSat‐null mice maintained on either low‐fat or high‐fat diets gain weight and have similar rates of food intake as age‐ and gender‐matched wildtype control littermates. This increased adiposity of RetSat‐null mice is associated with up‐regulation of PPARγ, a key transcriptional regulator of adipogenesis, and also its downstream target, fatty acid‐binding protein 4 (FABP4/aP2). On the basis of these results, we propose that dihydroretinoids produced byRetSat control physiological processes that influence PPARγ activity and regulate lipid accumulation in mice.—Moise, A. R., Lobo, G. P., Erokwu, B., Wilson, D. L., Peck, D., Alvarez, S., Domínguez, M., Alvarez, R., Flask, C. A., de Lera, A. R., von Lintig, J., Palczewski, K. Increased adiposity in the retinol saturase‐knockout mouse. FASEB J. 24, 1261–1270 (2010). www.fasebj.org

Combining the Antigen Processing Components TAP and Tapasin Elicits Enhanced Tumor-Free Survival

Purpose: Tpn is a member of the MHC class I loading complex and functions to bridge the TAP peptide transporter to MHC class I molecules. Metastatic human carcinomas often express low levels of the antigen-processing components Tapasin and TAP and display few functional surface MHC class I molecules. As a result, carcinomas are unrecognizable by effector CTLs. The aim of this study is to examine if Tapasin (Tpn) plays a critical role in the escape of tumors from immunologic recognition. Experimental Design: To test our hypothesis, a nonreplicating adenovirus vector encoding human Tpn (AdhTpn) was constructed to restore Tpn expression in vitro and in vivo in a murine lung carcinoma cell line (CMT.64) that is characterized by down-regulation of surface MHC class I due to deficiency in antigen-processing components. Results:Ex vivo, Tpn expression increased surface MHC class I and restored susceptibility of tumor cells to antigen-specific CTL killing, and AdhTpn infection of dendritic cells also significantly increased cross-presentation and cross-priming. Furthermore, tumor-bearing animals inoculated with AdhTpn demonstrated a significant increase in CD8+ and CD4+ T cells and CD11c+ dendritic cells infiltrating the tumors. Provocatively, whereas syngeneic mice bearing tumors that were inoculated with AdhTpn a significant reduction in tumor growth and increased survival compared with vector controls, combining AdhTpn inoculation with AdhTAP1 resulted in a significant augmentation of protection from tumor-induced death than either component alone. Conclusions: This is the first demonstration that Tpn alone can enhance survival and immunity against tumors but additionally suggests that Tpn and TAP should be used together as components of immunotherapeutic vaccine protocols to eradicate tumors.