Mycobacterium tuberculosis infection is exacerbated in mice lacking lecithin:retinol acyltransferase.
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ABSTRACT:
SUBMITTER: Trasino SE
PROVIDER: S-EPMC7484280 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We analyzed the retinoid levels and gene expression in various tissues after wild-type (Wt) and lecithin:retinol acyltransferase (LRAT-/-) knockout mice were fed a high retinol diet (250 IU/g). As compared to Wt, LRAT-/- mice exhibited a greater and faster increase in serum retinol concentration (mean+/-S.D., Wt, 1.3 +/- 0.2 microM to 1.5 +/- 0.3 microM in 48 h, p > 0.05; LRAT-/-, 1.3 +/- 0.2 microM to 2.2+/-0.3 microM in 48 h, p < 0.01) and a higher level of retinol in adipose tissue (17.2 +/- 2.4 pmol/mg in Wt vs. 34.6 +/- 8.0 pmol/mg in LRAT-/-). In the small intestines of Wt mice higher levels of retinol (96.4 +/- 13.0 pmol/mg in Wt vs. 13.7 +/- 7.6 pmol/mg in LRAT-/- and retinyl esters (2493.4 +/- 544.8 pmol/mg in Wt vs. 8.2 +/- 2.6 pmol/mg in LRAT-/- were detected. More retinol was detected in the feces of LRAT-/- mice (69.3 +/- 32.6 pmol/mg in LRAT-/- vs. 24.1 +/- 8.6 pmol/mg in Wt). LRAT mRNA levels increased in the lungs, small intestines, and livers of Wt mice on the high retinol diet, while CYP26A1 mRNA levels increased greatly only in the LRAT-/- mice. After 4 weeks, no significant differences between Wt mice and LRAT-/- mice were observed in either the serum retinol level or in the prevalence of Goblet cells in jejunal crypts. Our data indicate that the LRAT-/- mice maintain the homeostasis of retinol as the dietary retinol increases by increasing the excretion of retinol from the gastrointestinal tract, increasing the distribution of retinol to adipose tissue, and enhancing the catabolism by CYP26A1. We show that LRAT plays a role in maintaining a stable serum retinol concentration when dietary retinol concentration fluctuates.
| S-EPMC2486431 | biostudies-literature
Project description:Membrane-bound lecithin retinol acyltransferase (LRAT), an essential enzyme in vitamin A processing, catalyzes the formation of retinyl esters from vitamin A and lecithin. Cloned and expressed LRAT has a molecular mass of 25.3 kDa. The enzyme is not homologous to known enzymes and is, therefore, of substantial interest mechanistically. Along these lines, the functional protomeric state of LRAT is of importance. Gel electrophoretic studies on LRAT in the presence of SDS and disulfide reducing agents show the expected 25 kDa monomer. However, gel electrophoresis in the absence of a reducing agent and/or strong denaturing conditions reveals substantial dimer formation. LRAT monomers can be efficiently and irreversibly cross-linked by thiol reactive bismaleimides in retinal pigment epithelial (RPE) membranes generating LRAT homodimers. Cross-linked LRAT homodimers are fully active catalytically. The experiments suggest that LRAT monomers interact in membranes and form functional homodimers through protein-protein interactions and disulfide bond formation.
| S-EPMC5503676 | biostudies-literature
Project description:Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes.
| S-EPMC3390653 | biostudies-literature
Project description:Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys(161) residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.
| S-EPMC2937952 | biostudies-literature
Project description:Lecithin:retinol acyltransferase (LRAT) regulates retinol (vitamin A) metabolism by esterifying retinol. LRAT expression is decreased in cultured human squamous cell carcinoma cells of the head and neck relative to normal epithelial cells. We investigated whether the carcinogen 4-nitroquinoline 1-oxide (4-NQO) induced a higher incidence of oral cancer in LRAT knockout (LRAT(-/-)) than in wild-type (Wt) mice. We also investigated retinol deprivation during 4-NQO treatment in LRAT(-/-) mice as a model for rapid retinol deficiency. We observed higher levels of secreted frizzled-related protein (Sfrp) 2, an inhibitor of WNT signaling, in tongue tumors in LRAT(-/-) versus Wt. LRAT(-/-) embryonic stem cells also expressed higher Sfrp2 transcripts, indicating an interaction between retinol and WNT signaling. Cox-2, Cyclin D1, p21, Trop2 and RARβ2 were not differentially expressed in Wt versus LRAT(-/-) tongue tumors. Wt and LRAT(-/-) mice fed a retinol-sufficient diet showed the same oral tumor incidence after 4-NQO treatment. In contrast, tongue tumors developed in 60% of Wt mice and in 100% of LRAT(-/-) mice fed a retinol-deficient diet during 4-NQO treatment (P=.22); moreover, the bromodeoxyuridine labeling index was 21.0 ± 2.4% in LRAT(-/-) normal tongue epithelium as compared to 9.9 ± 0.8% in Wt normal tongue epithelium (P<.001). Thus, partial retinol deficiency during carcinogen treatment (achieved in LRAT(-/-)) resulted in more proliferating cells in tongue epithelia from LRAT(-/-) mice and, ultimately, a greater probability of carcinogenesis.
| S-EPMC2941705 | biostudies-literature
Project description:Lecithin:retinol acyltransferase (LRAT) catalyzes the acyl transfer from the sn-1 position of phosphatidylcholine (PC) to all-trans-retinol, creating fatty acid retinyl esters (palmitoyl, stearoyl, and some unsaturated derivatives). In the eye, these retinyl esters are substrates for the 65 kDa retinoid isomerase (RPE65). LRAT is well characterized biochemically, and recent structural data from closely related family members of the NlpC/P60 superfamily and a chimeric protein have established its catalytic mechanism. Mutations in the LRAT gene are responsible for approximately 1% of reported cases of Leber congenital amaurosis (LCA). Lack of functional LRAT, expressed in the retinal pigmented epithelium (RPE), results in loss of the visual chromophore and photoreceptor degeneration. LCA is a rare hereditary retinal dystrophy with an early onset associated with mutations in one of 21 known genes. Protocols have been devised to identify therapeutics that compensate for mutations in RPE65, also associated with LCA. The same protocols can be adapted to combat dystrophies associated with LRAT. Improvement in the visual function of clinical recipients of therapy with recombinant adeno-associated virus (rAAV) vectors incorporating the RPE65 gene provides a proof of concept for LRAT, which functions in the same cell type and metabolic pathway as RPE65. In parallel, a clinical trial that employs oral 9-cis-retinyl acetate to replace the missing chromophore in RPE65 and LRAT causative disease has proven to be effective and free of adverse effects. This article summarizes the biochemistry of LRAT and examines chromophore replacement as a treatment for LCA caused by LRAT mutations.
| S-EPMC5555363 | biostudies-literature
Project description:Esterification of all-trans-retinol is a key reaction of the vertebrate visual cycle, since it produces an insoluble, relatively non-toxic, form of the vitamin for storage and supplies substrate for the isomerization reaction. CoA-dependent and -independent pathways have been described for retinol esterification in retinal pigment epithelium (RPE). The CoA-independent reaction, catalysed by lecithin:retinol acyltransferase (LRAT) was examined in more detail in this study. Addition of retinol to RPE microsomes results in a burst of retinyl ester synthesis, followed by a rapid apparent cessation of the reaction. However, [3H]retinol, added when retinyl ester synthesis has apparently ceased, is rapidly incorporated into retinyl ester without a net increase in the amount of ester. The specific radioactivities of [3H]retinol and [3H]retinyl ester reach the same value. [14C]Palmitate from palmitoyl-CoA is incorporated into preexisting retinyl ester in the absence of net ester synthesis, too. These exchange reactions suggest that the reaction has reached equilibrium at the plateau of the progress curve and that only the accumulation of retinyl ester, and not its synthesis, has stopped during this phase of the reaction. Studies with geometrical isomers of retinol revealed that the rate of exchange of all-trans-retinol with all-trans-retinyl esters was about 6 times more rapid than exchange of 11-cis-retinol with 11-cis-retinyl ester. This is the first demonstration of the reversibility of LRAT and the first example of stereospecificity of retinyl ester synthesis in the visual system. Reversal of the LRAT reaction could contribute to the mobilization of 11-cis-retinol from 11-cis-retinyl ester pools.
| S-EPMC1132424 | biostudies-other
Project description:Lecithin retinol acyltransferase (LRAT) catalyzes the reversible esterification of vitamin A using lecithin as the acyl donor. LRAT is the founder member of a new class of enzymes, which include class II tumor suppressors, proteins essential for development, and putative proteases. All of these proteins possess Cys and His residues homologous to C161 and H60 of LRAT. These two residues are shown here to be essential for LRAT activity and are part of a catalytic dyad reminiscent of that found in thiol proteases. However, the local primary sequence contexts of C161 and H60 of LRAT and family are not at all homologous to those found in the approximately 20 thiol protease families. Moreover, LRAT shows pKs of 8.3 and 10.8, compared to approximately 4.0 and 8.5 observed in the thiol proteases. LRAT also contains Gln177 and Asp67 residues, which are largely conserved in the homologues. However, neither of these residues is essential for catalysis. Thiol proteases often contain catalytically essential Asp or Gln residues. It is concluded that LRAT is the founder member of a new class of Cys-His enzymes with diverse functions.
| S-EPMC5511752 | biostudies-literature
Project description:Lecithin retinol acyltransferase (LRAT) catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester, an essential reaction in the vertebrate visual cycle. Since all-trans-retinyl esters are the substrates for the isomerization reaction that generates 11-cis-retinoids, this esterification reaction is essential in the operation of the visual cycle. In addition, LRAT is the founder member of a series of proteins, which are of novel sequence and have unknown functions. Native LRAT is an integral membrane protein and has never been purified. To obtain a pure LRAT, the N- and C-transmembrane termini were deleted and replaced with a poly His tag for the purpose of purification. This truncated form of LRAT, referred to as tLRAT, has been expressed in bacteria and fully purified. tLRAT is catalytically active and processes all-trans-retinol at least 10-fold more efficiently than 11-cis-retinol, the precursor to the visual chromophore. While tLRAT can be robustly expressed in bacteria, it requires detergent for extraction, as the enzyme still contains hydrophobic domains, which may interact. Indeed, tLRAT can oligomerize and forms dimers. Native LRAT also forms functional homodimers. These studies pave the way for the preparation of large-scale amounts of pure tLRAT for further mechanistic and structural studies.
| S-EPMC5507194 | biostudies-literature
Project description:Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.
| S-EPMC3271263 | biostudies-literature