Retinoic Acid Binding Protein Research Articles

Development of an Enzyme-Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly.

This paper presents an enzyme building block for the assembly of megamolecules. The system is based on the inhibition of the human-derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate group, which uses sulfur fluoride exchange click chemistry to covalently inhibit CRABP2. We conjugated both the inhibitor and a fluorescein tag to an oligo(ethylene glycol) backbone and measured a second-order rate constant for the protein inhibition reaction of approximately 3,600 M-1 s-1 . We used this new enzyme-inhibitor pair to assemble multi-protein structures in one-pot reactions using three orthogonal assembly chemistries to demonstrate exact control over the placement of protein domains within a single, homogeneous molecule. This work enables a new dimension of control over specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures.

Crabp1 Modulates HPA Axis Homeostasis and Anxiety-like Behaviors by Altering FKBP5 Expression.

Retinoic acid (RA), the principal active metabolite of vitamin A, is known to be involved in stress-related disorders. However, its mechanism of action in this regard remains unclear. This study reports that, in mice, endogenous cellular RA binding protein 1 (Crabp1) is highly expressed in the hypothalamus and pituitary glands. Crabp1 knockout (CKO) mice exhibit reduced anxiety-like behaviors accompanied by a lowered stress induced-corticosterone level. Furthermore, CRH/DEX tests show an increased sensitivity (hypersensitivity) of their feedback inhibition in the hypothalamic–pituitary–adrenal (HPA) axis. Gene expression studies show reduced FKBP5 expression in CKO mice; this would decrease the suppression of glucocorticoid receptor (GR) signaling thereby enhancing their feedback inhibition, consistent with their dampened corticosterone level and anxiety-like behaviors upon stress induction. In AtT20, a pituitary gland adenoma cell line elevating or reducing Crabp1 level correspondingly increases or decreases FKBP5 expression, and its endogenous Crabp1 level is elevated by GR agonist dexamethasone or RA treatment. This study shows, for the first time, that Crabp1 regulates feedback inhibition of the the HPA axis by modulating FKBP5 expression. Furthermore, RA and stress can increase Crabp1 level, which would up-regulate FKBP5 thereby de-sensitizing feedback inhibition of HPA axis (by decreasing GR signaling) and increasing the risk of stress-related disorders.

Nuclear factor I/X (NFIX) regulates the transcriptional activity of the cellular retinoic acid binding protein 2 (CRABP2) promoter and alters CRABP2 expression in Marshall-Smith Syndrome (MSS) patients.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid–binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the kcat/Km values either decreased 5-fold or were equal to the respective free retinoid values. The kcat/Km value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in kcat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.

Estrogen regulates the expression of retinoic acid synthesis enzymes and binding proteins in mouse skin

Topical 17-beta-estradiol (E2) regulates the hair cycle, hair shaft differentiation, and sebum production. Vitamin A also regulates sebum production. Vitamin A metabolism proteins localized to the pilosebaceous unit (PSU; hair follicle and sebaceous gland); and were regulated by E2 in other tissues. This study tests the hypothesis that E2 also regulates vitamin A metabolism in the PSU. First, aromatase and estrogen receptors localized to similar sites as retinoid metabolism proteins during mid-anagen. Next, female and male wax stripped C57BL/6J mice were topically treated with E2, the estrogen receptor antagonist ICI 182,780 (ICI), letrozole, E2 plus letrozole, or vehicle control (acetone) during mid-anagen. E2 or one of its inhibitors regulated most of the vitamin A metabolism genes and proteins examined in a sex-dependent manner. Most components were higher in females and reduced with ICI in females. ICI reductions occurred in the premedulla, sebaceous gland, and epidermis. Reduced E2 also reduced RA receptors in the sebaceous gland and bulge in females. However, reduced E2 increased the number of retinal dehydrogenase 2 positive hair follicle associated dermal dendritic cells in males. These results suggest that estrogen regulates vitamin A metabolism in the skin. Interactions between E2 and vitamin A have implications in acne treatment, hair loss, and skin immunity.

Abstract 658: CRABP2 - a novel biomarker for high risk endometrial cancer

Abstract Introduction: The incidence of endometrial cancer (EC) is rising - a consequence of the current obesity epidemic. An improved understanding of obesity-induced EC is needed to manage this growing cohort of patients. Here-in we present Cellular Retinoic Acid Binding Protein 2 (CRABP2), which was identified as an obesity-related gene in the BDII/Han rat spontaneous EC model, as a novel biomarker in high-risk EC. Methods:BDII/Han rats were fed either a high (N=12) or low calorie (N=7) diet for 15 months. RNA-seq, - differential gene expression and gene set enrichment analysis (GSEA) were performed on endometrial tumors from both groups. The association between CRABP2 mRNA expression and standard clinico-pathological features was examined in human EC using the TCGA. Following dichotomization of patients based on their CRABP2 mRNA expression using regression tree analysis, univariate and multivariate Cox regression survival analyses were performed. Differential gene expression analysis and GSEA were performed on human tumors stratified according to CRABP2 expression levels. In-silico findings were validated using immunohistochemistry (IHC) assessing CRABP2 expression in normal endometrium, primary and metastatic endometrial carcinoma. Results:Weight gain in high fat diet (HFD) rats was double that of normal chow diet rats (50 g vs. 25 g). The incidence of cancer was similar in both groups (4/7-57% vs. 4/12-33%; p=0.37). 368 differentially expressed genes (DEGs) were identified between both groups. CRABP2 was upregulated in tumors from the HFD group. Regression tree analysis identified 60 (11%) tumors in the TCGA with increased CRABP2 mRNA expression. Increased CRABP2 expression was associated with serous EC (p<0.001), grade 3 (p<0.001) and advanced stage (p=0.015). Univariate Cox regression analysis revealed that high CRABP2 expression is associated with reduced overall survival (HR=2.67, 95% CI 1.64-4.33, p<0.001). This remained true following multivariate cox regression analysis in which age, histological type, stage and grade were confounded for (HR=2.06, 95% CI 1.24-3.41, p=0.004). 224 DEGs were identified between CRABP2 high (N=60) and low (N=468) tumors in the TCGA. GSEA analysis revealed an enrichment of pathways involved in developmental biology and embryonic stem cell-like gene expression signatures in the CRABP2-high cohort. IHC confirmed increased expression of cytoplasmic CRABP2 in EC compared to normal endometrium. CRABP2 expression was significantly increased in serous compared to endometrioid cancer (p<0.05) and a comparison between primary and metastatic serous carcinoma demonstrated increased expression in metastatic cases. Conclusion: A HFD increased CRABP2 expression in rat EC. In humans, increased expression of CRABP2 is associated with high-risk EC. Transcriptomic analysis and validation using IHC suggests that CRABP2 and retinoic acid signalling may be an important therapeutic target in advanced EC. Citation Format: Donagh Egan, Bruce Moran, Michael Wilkinson, William Gallagher, Xavier Matias-Guiu, Walter Kolch, Carel LeRoux, Donal Brennan. CRABP2 - a novel biomarker for high risk endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 658.

Abstract 1401: MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers

Abstract Introduction: HER2-positive (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. The development of a targeted drug, trastuzumab, has greatly improved clinical outcome for HER2+ patients. However, intrinsic and acquired resistance to trastuzumab are common. Clearly, additional therapeutic interventions are required for the clinical management of HER2-enriched breast cancers. Retinoic acid (RA) is a vital signaling molecule that regulates multiple biological processes, including cell proliferation, differentiation, and death. RA has been successfully used in the treatment of acute promyelocytic leukemia. RA also holds promise for the treatment of solid cancers like breast cancer. RA action is mediated through transcription factor RARα and the RA binding protein CRABP2, which deliver RA to RARα in the nucleus. The HER2 gene (ERBB2) is frequently co-amplified with the gene encoding RARα, a key determinant of RA sensitivity. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. MYC is an oncogene that inhibits RARα activity in leukemia cells. Importantly, MYC is preferentially amplified and overexpressed in HER2+ breast cancers. My research aims to elucidate the mechanism underlying RA resistance in HER2+ breast cancers, with a special focus on the role of MYC. Hypothesis: MYC attenuates RA action by inhibiting the CRABP2-RARα pathway. Results: RNA levels of MYC are negatively correlated with CRABP2 RNA levels. Depletion of MYC upregulates CRABP2 at both the RNA and protein levels. Furthermore, we found that MYC binds to the CRABP2 promoter region suggesting that MYC may directly suppress CRABP2 gene transcription activity. Our results show that ectopic expression of MYC inhibits, whereas depletion of MYC activates RAR activity. Consistently, ectopic expression of MYC increases RA resistance, whereas depletion of MYC sensitizes cells to RA treatment. When CRABP2 is depleted along with MYC-knockdown, cell proliferation is rescued, suggesting that MYC mediates RA resistance, at least partially through downregulation of CRABP2. We also found that RA treatment enhances trastuzumab responsiveness in HER2+ breast cancer cells. Significance: This study sheds light on the role and mechanism of MYC in governing RA resistance in HER2+ breast cancer cells. Our results support the use of RA and trastuzumab for the treatment of subsets of patients with HER2+/low MYC breast cancers. Citation Format: Won-Shik Choi, Rong-Zong Liu, Roseline Godbout. MYC mediates retinoic acid resistance by suppressing cellular retinoic acid-binding protein (CRABP2) transcription in HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1401.

Abstract 2662: Cellular retinoic acid-binding protein 2 enhances saturated fatty acid-induced prostate cancer progression

Abstract Introduction: Epidemiological evidence suggests that saturated fatty acid has a role in prostate cancer progression; however underlying mechanisms remain unknown. Here, we conducted a comprehensive omics analysis to investigate candidate molecules associated with saturated fat-induced prostate cancer (PCa) progression. Methods: TRAMP-C2 cells were subcutaneously injected into two groups of C57BL6 mice: the lard diet (LD) group, which was fed isocaloric saturated fatty acid, and the fish oil diet (FOD) group, which was fed polyunsaturated fatty acid. We performed an oligonucleotide microarray with the Affymetrix Gene Chip Mouse Gene 2.0 ST Array and proteomic analysis using nano-liquid chromatography-tandem mass spectrometry using xenograft tumors and validated the result by western blotting and immunohistochemistry. The cell proliferation and mRNA levels of CRABP2 in two PCa cell lines cultured with mice serum were assessed by performing an MTT cell assay and quantitative real-time polymerase chain reaction. The effect of CRABP2 on survival in PCa patients was evaluated by reviewing data from The Cancer Genome Atlas database. Results: The mean body weight of the LD group tend to be higher than that of the LD group (p = 0.066). Subcutaneous tumor volume were significantly higher in the LD group than the FOD group (p = 0.044). One hundred fifty-three up- and 120 downregulated genes and 734 up- and 1514 downregulated proteins with >1.5-fold changes were identified in the LD group. Multi-omics approach showed 32 candidate molecules which have the potential to be highly associated with saturated fatty acid diet-induced prostate cancer progression. In the molecules, CRABP2 was selected as the most candidate molecule. The higher expression of CRABP2 in xenografts of the LD group was validated by western blotting and immunohistochemistry. PCa cells cultured in a medium containing LD mouse serum were associated with significantly higher cell proliferation rates and higher CRABP2 expression levels than those of PCa cells cultured in FOD. Overall survival significantly worse in the patients with altered CRABP2 than in those with unaltered CRABP2 (p < 0.001). Conclusion: Saturated fatty acid-accelerated PCa growth was enhanced by CRABP2. CRABP2 may be a key regulator of diet-induced PCa progression and a novel therapeutic target for PCa. A low saturated fat diet in males may be encouraged. Citation Format: Hiromi Sato, Shintarao Narita, Masanori Ishida, Soki Kashima, Ryohei Yamamoto, Atsushi Koizumi, Taketoshi Nara, Kazuyuki Numakura, Mitsuru Saito, Shigeru Satoh, Naoshi Dohmae, Tomonori Habuchi. Cellular retinoic acid-binding protein 2 enhances saturated fatty acid-induced prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2662.

Regulation of exosome secretion by cellular retinoic acid binding protein 1 contributes to systemic anti-inflammation

BackgroundIntercellular communications are important for maintaining normal physiological processes. An important intercellular communication is mediated by the exchange of membrane-enclosed extracellular vesicles. Among various vesicles, exosomes can be detected in a wide variety of biological systems, but the regulation and biological implication of exosome secretion/uptake remains largely unclear.MethodsCellular retinoic acid (RA) binding protein 1 (Crabp1) knockout (CKO) mice were used for in vivo studies. Extracellular exosomes were monitored in CKO mice and relevant cell cultures including embryonic stem cell (CJ7), macrophage (Raw 264.7) and hippocampal cell (HT22) using Western blot and flow cytometry. Receptor Interacting Protein 140 (RIP140) was depleted by Crispr/Cas9-mediated gene editing. Anti-inflammatory maker was analyzed using qRT-PCR. Clinical relevance was accessed by mining multiple clinical datasets.ResultsThis study uncovers Crabp1 as a negative regulator of exosome secretion from neurons. Specifically, RIP140, a pro-inflammatory regulator, can be transferred from neurons, via Crabp1-regulated exosome secretion, into macrophages to promote their inflammatory polarization. Consistently, CKO mice, defected in the negative control of exosome secretion, have significantly elevated RIP140-containing exosomes in their blood and cerebrospinal fluid, and exhibit an increased vulnerability to systemic inflammation. Clinical relevance of this pathway is supported by patients’ data of multiple inflammatory diseases. Further, the action of Crabp1 in regulating exosome secretion involves its ligand and is mediated by its downstream target, the MAPK signaling pathway.ConclusionsThis study presents the first evidence for the regulation of exosome secretion, which mediates intercellular communication, by RA-Crabp1 signaling. This novel mechanism can contribute to the control of systemic inflammation by transferring an inflammatory regulator, RIP140, between cells. This represents a new mechanism of vitamin A action that can modulate the homeostasis of system-wide innate immunity without involving gene regulation.EB6KXQYbkzhu3R444VcR3RVideo

Maprotiline Suppresses Cholesterol Biosynthesis and Hepatocellular Carcinoma Progression Through Direct Targeting of CRABP1.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related death and has a poor prognosis worldwide, thus, more effective drugs are urgently needed. In this article, a small molecule drug library composed of 1,056 approved medicines from the FDA was used to screen for anticancer drugs. The tetracyclic compound maprotiline, a highly selective noradrenergic reuptake blocker, has strong antidepressant efficacy. However, the anticancer effect of maprotiline remains unclear. Here, we investigated the anticancer potential of maprotiline in the HCC cell lines Huh7 and HepG2. We found that maprotiline not only significantly restrained cell proliferation, colony formation and metastasis in vitro but also exerted antitumor effects in vivo. In addition to the antitumor effect alone, maprotiline could also enhance the sensitivity of HCC cells to sorafenib. The depth studies revealed that maprotiline substantially decreased the phosphorylation of sterol regulatory element-binding protein 2 (SREBP2) through the ERK signaling pathway, which resulted in decreased cholesterol biosynthesis and eventually impeded HCC cell growth. Furthermore, we identified cellular retinoic acid binding protein 1 (CRABP1) as a direct target of maprotiline. In conclusion, our study provided the first evidence showing that maprotiline could attenuate cholesterol biosynthesis to inhibit the proliferation and metastasis of HCC cells through the ERK-SREBP2 signaling pathway by directly binding to CRABP1, which supports the strategy of repurposing maprotiline in the treatment of HCC.

Overexpression of CRABP2 inhibits dexamethasone-induced apoptosis in human osteoblast cells

BackgroundThe purpose of the current study was to explore the role and underlying mechanism of cellular retinoic acid binding protein 2 (CRABP2) in dexamethasone (DEX)-induced apoptosis in human osteoblast cells.MethodsGSE10311 was downloaded from the Gene Expression Omnibus (GEO) database to identify the differentially expressed genes (DEGs) by the limma/R package. Primary human osteoblast was isolated and treated with different concentration of DEX (0, 10-8, 10-7, 10-6, 10-5, and 10-4 mol/L), and cell viability and flow cytometry were used to detect cell proliferation and apoptosis. A CRABP2 overexpression plasmid (oe-CRABP2) was used to overexpress CRABP2, and western blotting was conducted to detect protein expression.ResultsWe found that CRABP2 was downregulated in the DEX-treated group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that DEGs were associated with PI3K/Akt signaling pathway. DEX downregulated CRABP2 gene and protein expression, inhibited viability, and induced human osteoblast apoptosis. Overexpression of CRABP2 reversed DEX-induced apoptosis in human osteoblast. Moreover, overexpression of CRABP2 delayed the progression of DEX-induced osteonecrosis of the femoral head (ONFH) animal model.ConclusionIn conclusion, CRABP2 is effective at inhibiting DEX-induced human osteoblast apoptosis and delayed ONFH progression.

LINC01816 promotes the migration, invasion and epithelial‑mesenchymal transition of thyroid carcinoma cells by sponging miR‑34c‑5p and regulating CRABP2 expression levels.

Thyroid carcinoma (THCA) is a common type of endocrine system cancer and its current clinical treatment method is surgical resection. Long non-coding RNAs (lncRNAs) have been revealed to serve important roles in a variety of complex human diseases. Therefore, determining the association between lncRNAs and diseases may provide novel insight into disease-related lncRNAs, with the aim of improving disease treatments and diagnoses. Long intergenic non-protein coding RNA 1816 (LINC01816) was identified to be associated with the survival of patients with colorectal cancer using the IDHI-MIRW method. The present study aimed to investigate the role and molecular mechanism of LINC01816 in THCA. Analysis of datasets from The Cancer Genome Atlas database revealed that the upregulation of LINC01816 expression levels was associated with a variety of cancer types. Reverse transcription-quantitative PCR analysis demonstrated that compared with the normal thyroid tissues, the expression levels of LINC01816 were upregulated in THCA tissues. The results of wound healing and Transwell assays, and western blotting demonstrated that the overexpression of LINC01816 could strengthen the invasive and migratory abilities of THCA cells and enhance epithelial-mesenchymal transition progression. Analysis using the starBase website and dual-luciferase reporter assays identified that microRNA (miR)-34c-5p was a target of LINC01816. The overexpression of miR-34c-5p could inhibit the invasive and migratory abilities of THCA cells, in addition to inhibiting the cellular retinoic acid binding protein 2 (CRABP2) overexpression-induced effects on invasion, migration and EMT processes. In conclusion, the findings of the present study indicated that LINC01816 may be capable of sponging miR-34c-5p to upregulate CRABP2 expression levels, which subsequently promoted the invasion, migration and EMT of THCA cells. Therefore, targeting the LINC01816/miR-34c-5p/CRABP2 pathway may be an effective therapeutic approach for patients with THCA.

Polyethylene glycol perturbs the unfolding of CRABP I: A correlation between experimental and theoretical approach

The importance of macromolecules paves the way towards a detailed molecular level investigation as all most all cellular processes occurring at the interior of cells in the form of proteins, enzymes, and other biological molecules are significantly affected because of their crowding. Thus, exploring the role of crowding environment on the denaturation and renaturation kinetics of protein molecules is of great importance. Here, CRABP I (cellular retinoic acid binding protein I) is employed as a model protein along with different molecular weights of Polyethylene glycol (PEG) as molecular crowders. The experimental evaluations are done by accessing the protein secondary structure analysis using circular dichroism (CD) spectroscopy and unfolding kinetics using intrinsic fluorescence of CRABP I at 37 °C to mimic the in vivo crowding environment. The unfolding kinetics results indicated that both PEG 2000 and PEG 4000 act as stabilizers by retarding the unfolding kinetic rates. Both kinetic and stability outcomes presented the importance of crowding environment on stability and kinetics of CRABP I. The molecular dynamics (MD) studies revealed that thirteen PEG 2000 molecules assembled during the 500 ns simulation, which increases the stability and percentage of β-sheet. The experimental findings are well supported by the molecular dynamics simulation results.

Dietary Vitamin A Impacts Refractory Telogen.

Hair follicles cycle through periods of growth (anagen), regression (catagen), rest (telogen), and release (exogen). Telogen is further divided into refractory and competent telogen based on expression of bone morphogenetic protein 4 (BMP4) and wingless-related MMTV integration site 7A (WNT7A). During refractory telogen hair follicle stem cells (HFSC) are inhibited. Retinoic acid synthesis proteins localized to the hair follicle and this localization pattern changed throughout the hair cycle. In addition, excess retinyl esters arrested hair follicles in telogen. The purpose of this study was to further define these hair cycle changes. BMP4 and WNT7A expression was also used to distinguish refractory from competent telogen in C57BL/6J mice fed different levels of retinyl esters from two previous studies. These two studies produced opposite results; and differed in the amount of retinyl esters the dams consumed and the age of the mice when the different diet began. There were a greater percentage of hair follicles in refractory telogen both when mice were bred on an unpurified diet containing copious levels of retinyl esters (study 1) and consumed excess levels of retinyl esters starting at 12 weeks of age, as well as when mice were bred on a purified diet containing adequate levels of retinyl esters (study 2) and remained on this diet at 6 weeks of age. WNT7A expression was consistent with these results. Next, the localization of vitamin A metabolism proteins in the two stages of telogen was examined. Keratin 6 (KRT6) and cellular retinoic acid binding protein 2 (CRABP2) localized almost exclusively to refractory telogen hair follicles in study 1. However, KRT6 and CRABP2 localized to both competent and refractory telogen hair follicles in mice fed adequate and high levels of retinyl esters in study 2. In mice bred and fed an unpurified diet retinol dehydrogenase SDR16C5, retinal dehydrogenase 2 (ALDH1A2), and cytochrome p450 26B1 (CYP26B1), enzymes and proteins involved in RA metabolism, localized to BMP4 positive refractory telogen hair follicles. This suggests that vitamin A may contribute to the inhibition of HFSC during refractory telogen in a dose dependent manner.

Structure-functional relationship of cellular retinoic acid-binding proteins I and II interacting with natural and synthetic ligands.

A detailed understanding of the interactions between small-molecule ligands and their proposed binding targets is of the utmost importance for modern drug-development programs. Cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) facilitate a number of vital retinoid signalling pathways in mammalian cells and offer a gateway to manipulation of signalling that could potentially reduce phenotypes in serious diseases, including cancer and neurodegeneration. Although structurally very similar, the two proteins possess distinctly different biological functions, with their signalling influence being exerted through both genomic and nongenomic pathways. In this article, crystal structures are presented of the L29C mutant of Homo sapiens CRABPI in complex with naturally occurring fatty acids (1.64 Å resolution) and with the synthetic retinoid DC645 (2.41 Å resolution), and of CRABPII in complex with the ligands DC479 (1.80 Å resolution) and DC645 (1.71 Å resolution). DC645 and DC479 are two potential drug compounds identified in a recent synthetic retinoid development program. In particular, DC645 has recently been shown to have disease-modifying capabilities in neurodegenerative disease models by activating both genomic and nongenomic signalling pathways. These co-crystal structures demonstrate a canonical binding behaviour akin to that exhibited with all-trans-retinoic acid and help to explain how the compounds are able to exert an influence on part of the retinoid signalling cascade.

Correlation between Plasma Cellular Retinoic Acid-Binding Protein 2 and Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer Cells.

Our primary aim of the current study was to explore the correlation between plasma CRABP2 and migration, proliferation and invasion of non-small cell lung cancer (NSCLC) cells. Human lung cancer cell line A549 was used in the present study, which was cultured in 6-well plates (1 × 106 cells/well) and then transfected with pcDNA-CRABP2 and pcDNA, siRNA with the use of Lipofectamine 2000 based on the manufacturer's protocol. The expression of CRABP2 mRNA was detected through real-time PCR. Proliferation was further detected using MTT assays, and apoptosis was monitored and recorded with the application of flow cytometry. The expression of E-cadherin, MMP9, vimentin and related pathway proteins was detected by Western blotting assays. Transwell assays and cell scratch assays were utilized for the detection of migration and invasion ability of A549 cells. RT-PCR results showed The CRABP2 mRNA transcript levels in the CRABP2 overexpression group were higher when comparing those of the empty vector group (P < 0.05). By MTT assays, CRABP2 overexpression promoted cellular proliferation, while CRABP2 downregulation inhibited cellular proliferation. CRABP2 overexpression inhibited cell apoptosis and promoted cellular proliferation. The number of TUNEL staining positive cells was the lowest in the CRABP2 overexpression group, and the siRNA transfection group had increased apoptosis. CRABP2 downregulation reduced EMT in cells and cell migration and invasion reflected from western blotting results and cell migration and invasion assay results, respectively. Inhibition of plasma CRABP2 expression offers the potential in terms of reducing the expression of MAPKs and proteins in the NF-κB pathway and inhibiting the proliferation and migration of NSCLC cells, which is ideally suited for further treatment for NSCLC.

Single-cell transcriptomic analysis of small and large wounds reveals the distinct spatial organization of regenerative fibroblasts.

Wound‐induced hair follicle neogenesis (WIHN) has been an important model to study hair follicle regeneration during wound repair. However, the cellular and molecular components of the dermis that make large wounds more regenerative are not fully understood. Here, we compare and contrast recently published scRNA‐seq data of small scarring wounds to wounds that regenerate in hope to elucidate the role of fibroblasts lineages in WIHN. Our analysis revealed an over‐representation of the newly identified upper wound fibroblasts in regenerative wound conditions, which express the retinoic acid binding protein Crabp1. This regenerative cell type shares a similar gene signature to the murine papillary fibroblast lineage, which are necessary to support hair follicle morphogenesis and homeostasis. RNA velocity analysis comparing scarring and regenerating wounds revealed the divergent trajectories towards upper and lower wound fibroblasts and that the upper populations were closely associated with the specialized dermal papilla. We also provide analyses and explanation reconciling the inconsistency between the histological lineage tracing and the scRNA‐seq data from recent reports investigating large wounds. Finally, we performed a computational test to map the spatial location of upper wound fibroblasts in large wounds which revealed that upper peripheral fibroblasts might harbour equivalent regenerative competence as those in the centre. Overall, our scRNA‐seq reanalysis combining multiple samples suggests that upper wound fibroblasts are required for hair follicle regeneration and that papillary fibroblasts may migrate from the wound periphery to the centre during wound re‐epithelialization. Moreover, data from this publication are made available on our searchable web resource: https://skinregeneration.org/.

Microbial Production of Retinyl Palmitate and Its Application as a Cosmeceutical

Chemically synthesized retinyl palmitate has been widely used in the cosmetic and biotechnology industry. In this study, we aimed to demonstrate the microbial production of retinyl palmitate and the benefits of microbial retinyl palmitate in skin physiology. A heterologous retinyl palmitate biosynthesis pathway was reconstructed in metabolically engineered Escherichia coli using synthetic expression modules from Pantoea agglomerans, Salinibacter ruber, and Homo sapiens. High production of retinyl palmitate (69.96 ± 2.64 mg/L) was obtained using a fed-batch fermentation process. Moreover, application of purified microbial retinyl palmitate to human foreskin HS68 fibroblasts led to increased cellular retinoic acid-binding protein 2 (CRABP2) mRNA level [1.7-fold (p = 0.001) at 100 μg/mL], acceleration of cell proliferation, and enhancement of procollagen synthesis [111% (p < 0.05) at 100 μg/mL], strongly indicating an anti-ageing-related effect of this substance. These results would pave the way for large-scale production of retinyl palmitate in microbial systems and represent the first evidence for the application of microbial retinyl palmitate as a cosmeceutical.

Effect of micro-environment on protein conformation studied by fluorescence-based techniques

In the present work, the conformation of a model cytoplasmic protein, cellular retinoic acid-binding protein I (CRABP I), is investigated as a function of micro-environment in the terms of several anions, such as SO42−, HPO42−, and Cl− by employing fluorescence correlation spectroscopy (FCS) along with other biophysical techniques (fluorescence and circular dichroism (CD) spectroscopy). FCS observations revealed the impact of anions on the diffusion of protein as well as the hydrodynamic radius at room temperature. Fluorescence (steady-state and time-resolved lifetime) and CD results indicated the conformational change of the protein upon binding to different salts. Our results revealed the conformational changes of the protein during micro-environment conditions, which can be helpful to understand how cellular condition influences protein folding in vivo compared to in vitro folding.

Abstract 6141: Role of aldehyde dehydrogenase 1B1 in colon carcinogenesis in a newly developed mouse model

Abstract Introduction: Aldehyde dehydrogenases (ALDH), particularly ALDH1B1 has been shown to have increased expression in both human and APCMin mice colorectal tumors. Studies have proposed the use of ALDH1B1 as a potential novel human colorectal tumor marker. Previous in vitro studies have shown that ALDH1B1 plays a critical role in colon carcinogenesis. The goal of this study is to understand the in vivo role of ALDH1B1 in colon carcinogenesis using a newly developed mouse model, APCMinAldh1b1−/− mice, through characterization of its phenotype in tumor growth and development, and elucidating the underlying potential molecular mechanisms. Methods: We generated APCMinAldh1b1−/-. At 12-14 weeks old, the experimental group APCMinAldh1b1−/- (n=6) and control group APCMinAldh1b1+/+ (n=7) were euthanized. Tumor counts, size and burden for each intestinal segments were recorded. H&amp;E staining was performed and observed for tumor types and counts. Immunohistochemistry using the markers Ki-67 (proliferation), Cleave Caspase 3 (apoptosis), KRT20 (differentiation) were done in colon. IHC of retinoic acid binding proteins (anti-CRBP2 and anti-FABP5) were also performed Results: The experimental group had shown significantly higher total average intestinal tumor burden (6.8 fold) (p=0.0007) and total average tumor counts observed (3.6 fold) (p=0.02). In the colon, there was a 3.5-fold increase in average tumor burden (p=0.017) with a 2.7-fold increase in average tumor size (p=0.025)in the experimental group. We also noticed a trend of increased average tumor counts observed in the colon of the experimental group although statistically insignificant. Average tumor counts of ileum observed was 9-fold higher in experimental group with only 1 mice from control group showed observable small tumor growth which did not meet criteria for tumor size and tumor burden calculation. There were no significant differences in observable tumor counts, tumor size and tumor burden in jejunum. Histologically, all tumors were pedunculated adenocarcinomas. Many unobservable small tumors less than 1mm width and length were seen in the H&amp;E of both ileum and jejunum in the experimental and control group. In the control group, there was less observable tumors compared to the experimental group although the tumor counts on H&amp;E in both groups showed no major differences. This indicates that the tumor size in the experimental group was larger than the control group. On the contrary, average tumor counts of colon in H&amp;E were lower compared to tumor counts observed in both control and experimental groups, indicating low number of small unobservable tumors in colon. Although the tumor counts of colon on H&amp;E and observation between the two groups were statistically insignificant, we noticed a pattern of increased tumor counts in the experimental group suggesting a potential increase in tumor incidence. Immunohistochemical staining of colon is ongoing. Conclusion: ALDH1B1 reduced tumor growth rate in the ileum, and reduced tumor size in the colon suggesting its role in tumor promotion and progression. A larger number of experimental and control group is required to establish its potentially tumor-initiating role in the colon. Further characterization of ALDH1B1 in colon carcinogenesis is ongoing. We are also elucidating the role and regulation of retinoic acid signaling by ALDH1B1 in colorectal tumor growth. Citation Format: Wan Ying Tan, David. J. Orlicky, Ying Chen, Yatrik M. Shah, David C. Thompson, Vasilis Vasiliou. Role of aldehyde dehydrogenase 1B1 in colon carcinogenesis in a newly developed mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6141.