Vitamin E activates CRABP-II gene expression in cultured human fibroblasts, role of protein kinase C

The treatment of human fibroblasts with different tocopherols in the presence of retinol caused an increase in cytoplasmic retinoic acid binding protein II (CRABP-II) mRNA and protein. The possibility of an involvement of protein kinase C (PKC) in the response to tocopherols was supported by the results obtained with the PKC-specific inhibitors, calphostin C and bisindolylmaleimide I. The effect of alpha-tocopherol was prevented by okadaic acid, suggesting that a protein phosphatase is responsible for PKC dephosphorylation produced by the presence of tocopherols. The results shown support the hypothesis that phosphorylation/dephosphorylation of RXRalpha via PKC may be involved in the regulation of CRABP-II gene expression.     

Purification and partial characterization of a novel binding protein for retinoic acid from neonatal rat

This report describes the purification and partial characterization of a novel retinoic acid-binding protein (CRABP-II) from neonatal rat pups. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-cellulose, and high performance liquid chromatography (HPLC) on a DEAE 5PW column. Two retinoic acid-binding peaks were resolved at the DEAE-cellulose step, with CRABP-I in the major peak and CRABP-II in the minor peak. Apparent homogeneity was achieved for both binding proteins after the HPLC step. CRABP-II consists of a single polypeptide, migrating with an apparent Mr of 15,000 in sodium dodecyl sulfate-polyacrylamide gel. It has an isoelectric point of 5.0. The dissociation constant for CRABP-II of retinoic acid was estimated to be 65 nM by fluorescence titration. Amino-terminal sequence analysis showed that CRABP-II has a distinct sequence, while the CRABP-I sequence is exactly identical to that of the rat testis CRABP. Despite the extensive sequence homology between CRABP-I and CRABP-II, antibodies directed against CRABP-I did not cross-react with CRABP-II.     

Specific degradation of CRABP-II via cIAP1-mediated ubiquitylation induced by hybrid molecules that crosslink cIAP1 and the target protein

Manipulation of protein stability with small molecules is a challenge in the field of drug discovery. Here we show that cellular retinoic acid binding protein-II (CRABP-II) can be specifically degraded by a novel compound, SNIPER-4, consisting of (--)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-L-leucine methyl ester and all-trans retinoic acid that are ligands for cellular inhibitor of apoptosis protein 1 (cIAP1) and CRABP-II, respectively. Mechanistic analysis revealed that SNIPER-4 induces cIAP1-mediated ubiquitylation of CRABP-II, resulting in the proteasomal degradation. The protein knockdown strategy employing the structure of SNIPER-4 could be applicable to other target proteins.     

The level of CRABP-I expression influences the amounts and types of all-trans-retinoic acid metabolites in F9 teratocarcinoma stem cells.

The CRABP-I and CRABP-II proteins are high affinity cytoplasmic retinoic acid-binding proteins. In undifferentiated F9 teratocarcinoma stem cells, only the CRABP-I protein is expressed at detectable levels. We have previously shown that overexpression of the CRABP-I protein in stably transfected F9 stem cell lines results in a lower sensitivity to a given external concentration of retinoic acid relative to that of untransfected F9 cells; in contrast, reduced CRABP-I expression in CRABP-I cDNA anti-sense transfected lines is associated with increased sensitivity of these lines to retinoic acid. These three types of cell lines were cultured in the presence of 50 nM [3H]retinoic acid, and the metabolism of retinoic acid was followed over the next 24 h. The results demonstrate that CRABP-I has the ability to alter both the levels and types of RA metabolites produced in the cytoplasm of differentiating embryonic stem cells. Moreover, the level of CRABP-I determines the rate of RA metabolism to 4-oxo-RA such that the higher the CRABP-I level, the faster the metabolism of [3H]retinoic acid. This is the first reported connection between the level of CRABP-I expression and intracellular RA metabolism.     

Effect of retinoic acid on cAMP dependent protein phosphorylation in psoriatic fibroblasts

Retinoic acid treatment of psoriatic fibroblasts increases the activity of cyclic AMP dependent protein kinase. In this study we report that retinoic acid treatment of cultured psoriatic fibroblasts modifies their subsequent cAMP dependent protein phosphorylation. In the soluble fraction of normal fibroblasts cAMP clearly enhances the in vitro phosphorylation of proteins of MW 37,49,54,56,68,83 kD while retinoic acid treatment of the same cells results in a decrease of the cAMP dependent phosphorylation of the first five of the same proteins. In contrast, in psoriatic fibroblasts from psoriatic patients retinoic acid either has no effect or increases the cAMP dependent phosphorylation of some of these proteins. Moreover the phosphorylation of a protein of MW 54 kD, undetectable in untreated psoriatic cells, is more phosphorylated in the presence of cAMP after retinoic acid treatment. The appearance of this phosphorylated proteins is time dependent and dose dependent upon the addition of retinoic acid. These in vitro phosphorylation results suggest that retinoic acid treatment of psoriatic fibroblasts change the level of cAMP dependent phosphorylation of some cytosolic proteins. These specific phosphorylations could be implicated in a variation of cell functions.     

Identification of proteins differentially expressed during chondrogenesis of mesenchymal cells

We performed comparative proteome analysis of mesenchymal cells and chondrocytes to identify proteins differentially expressed during chondrogenesis. Nine such proteins were identified. Type II collagen, matrilin-1, carbonic anhydrase-II (CA-II), 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2, and aldo-keto reductase were increased during chondrogenesis, whereas cellular retinoic acid binding protein-I (CRABP-I), CRABP-II, cytoplasmic type 5 actin, and fatty acid binding protein were decreased or almost disappeared. Expression of type II collagen, matrilin-1, PAPS synthetase-2, and CA-II was regulated by extracellular signal-regulated protein kinase, protein kinase C, and p38 kinase, signaling molecules known to regulate chondrogenesis.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.     

A novel protein highly expressed in testis is overexpressed in systemic sclerosis fibroblasts and targeted by autoantibodies

Nearly all autoantibody specificities in sera from patients with systemic sclerosis (SSc) target proteins distributed ubiquitously, and Abs against proteins whose expression is restricted to the affected sites have not been identified. In this study we describe SSc-specific autoantibody to a novel testicular Ag, termed protein highly expressed in testis (PHET), which is ectopically overexpressed in SSc dermal fibroblasts. A partial cDNA encoding PHET was isolated by immunoscreening of a HepG2 cDNA library with an SSc serum. PHET appeared to be a member of the UniGene cluster Hs.129872, but had a unique exon composition and a characteristic mRNA expression profile restricted to the testis. Serum Abs to a recombinant PHET fragment were detected in nine (8.4%) of 107 SSc patients, but in none of 50 systemic lupus erythematosus patients or 77 healthy controls. In SSc patients, the presence of anti-PHET Abs was associated with diffuse cutaneous SSc and lung involvement (p = 0.02 and 0.01, respectively). PCR-based quantitative analysis of PHET mRNA expression in cultured dermal fibroblasts showed increased expression of PHET mRNA in SSc fibroblasts compared with control fibroblasts. PHET-reactive Abs purified from SSc sera stained the cytoplasm of SSc dermal fibroblasts, and the staining intensity tended to be more prominent on SSc compared with control fibroblasts. These findings suggest that the autoantibody response to PHET can be induced by ectopic overexpression of PHET in dermal fibroblasts in SSc patients.