Enzymes and binding proteins affecting retinoic acid concentrations

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alchol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low K_m substrate.     

Folding of intracellular retinol and retinoic acid binding proteins

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.     

A high-performance liquid chromatographic technique that separates cellular retinol binding protein from cellular retinoic acid binding protein

A one-step procedure to detect cellular [3H]retinol and [3H]retinoic acid binding proteins (CRBP and CRABP) from rat testis cytosolic extract was devised. The procedure is based on anion-exchange high-performance liquid chromatography of the cytosolic fraction on columns of Mono Q, which permits elution of CRABP and CRBP at 12 and 22 min, respectively.     

A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II

Primary sequences of proteins often contain motifs that serve as "signatures" for subcellular targeting, such as a nuclear localization signal (NLS). However, many nuclear proteins do not harbor a recognizable NLS, and the pathways that mediate their nuclear translocation are unknown. This work focuses on CRABP-II, a cytosolic protein that moves to the nucleus upon binding of retinoic acid. While CRABP-II does not contain an NLS in its primary sequence, such a motif could be recognized in the protein's tertiary structure. We map the retinoic acid-induced structural rearrangements that result in the presence of this NLS in holo- but not apo-CRABP-II. The signal, whose three-dimensional configuration aligns strikingly well with a "classical" NLS, mediates ligand-induced association of CRABP-II with importin alpha and is critical for nuclear localization of the protein. The ligand-controlled NLS "switch" of CRABP-II may represent a general mechanism for posttranslational regulation of the subcellular distribution of a protein.     

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [³H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [³H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

NMR study of the binding of all-trans-retinoic acid to type II human cellular retinoic acid binding protein.

Cellular RA binding proteins are thought to play important roles in the (RA), a hormonally active metabolite of vitamin A that has profound effects on cell growth, + differentiation and morphogenesis. Binding of RA to type II human cellular RA binding proteins (CRABPII) has been investigated by NMR spectroscopy. The sequential resonance assignments of +CRABPII in the presence of RA were established by heteronuclear three-dimensional NMR at pH 7.3. The resonance assignments of the bound RA were achieved by homonucl NMR. The secondary structures of holo-CRABPII determined by NMR were ess as revealed by the crystal structure of holo-CRABPII. Most of the nuclear Overhauser effects (NOEs) between CRABPII and the bound RA were consistent with those predicted crystal structure of holo-CRABPII. The results suggested that the conformations in solution and in the crystalline state are highly similar. Compared to the ligand binding pocket, especially the ligand entrance, was stabilize Ser12-Leu18, one of the structure elements that constitute the ligand binding pocket, became more mobile upon binding of RA. Intramolecular NOEs between protons of the bo the carboxylate end of the bound RA is well fixed but the β-ionone     

Expression of the cellular retinoic acid binding proteins,type II and type I,in mature rat olfactory epithelium

Cellular retinoic acid binding proteins, types I and II (CRABP I and II), are cytosolic proteins that exhibit a binding preference for all-trans retinoic acid. As part of a larger study to determine whether retinoic acid plays a role in neurogenesis in vivo, we questioned whether CRABP II is present in rat postnatal olfactory epithelium (OE), a sensory tissue that continually replaces neurons throughout adult life. We have determined that both CRABP II and CRABP I proteins and the mRNAs that encode them are present in postnatal rat OE. Immunoreactivity with CRABP II and CRABP I antibodies was not observed in the nasal respiratory epithelium. Double immunolabeling experiments, conducted with antibodies showing specificity for each antigen, indicate that CRABP II and CRABP I are found in different cell types within the olfactory neuroepithelium. We also asked whether CRABP II is expressed in the postnatal rat retina, a neural tissue that is not known to show neuron replacement during adult life. CRABP type II immunoreactivity was not observed in the mature rat retina. The presence of CRABP II in postnatal OE and its absence from mature retina is consistent with previous reports indicating that the distribution of CRABP II in adult mammals is restricted to tissue systems that exhibit ongoing growth and differentiation throughout life.     

Separable binding proteins for retinoic acid and retinol in bovine retina.

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Saturation analysis of cellular retinoid binding proteins: application to retinoic acid resistant human neuroblastoma cells and to human tumors

A method for saturation analysis of cellular retinoic acid and retinol binding proteins, CRABP and CRBP, respectively, in cultured cells and human tumor samples, and its application to a retinoic acid resistant subline of the human neuroblastoma LA-N-5 cell line is described. Assessment of retinoid binding was accomplished by incubation of cytosols with increasing concentrations of [3H]retinoid (28-43 Ci/mmol; 1 Ci = 37 GBq) for 24 h. Bound retinoid was separated from free retinoid by adsorption with dextran-coated charcoal. Nonspecific binding was quantitated in parallel incubations which had been treated with p-chloromercuribenzene sulfonate (PCMBS), resulting in selective elimination of sulfhydryl-dependent ligand binding to both CRABP and CRBP. Quantitation was accomplished by Scatchard analysis of specific (PCMBS sensitive) binding. Employing this technique, specific retinoid binding was attributed to the presence of 2S macromolecules which displayed the known properties of CRABP and CRBP, namely ligand specificity, saturability, high ligand affinity, and PCMBS sensitivity. The apparent dissociation constants (Kd) for retinoic acid binding in cytosols prepared from murine 3T6 fibroblasts, rat testes, and a human ovarian tumor were 7, 11, and 35 nM, respectively. These preparations also bound retinol with high affinity, exhibiting Kds of 12, 26, and 48 nM, respectively. A retinoic acid resistant subline of LA-N-5 cells designated LA-N-5-R9 was established by long-term culture in the presence of 10(-6) M retinoic acid. This subline is resistant to the effects of retinoic acid in that it requires a 10-fold higher concentration of retinoic acid for 50% inhibition of growth than the parent line and displays no retinoic acid induced morphologic differentiation. Saturation analysis of CRABP in the parent and resistant subline reveal no significant alteration in either CRABP content or affinity. These results indicate that resistance to retinoic acid induced differentiation in LA-N-5-R9 occurs distal to CRABP binding or that CRABP does not mediate this response to retinoic acid.     

Dissection of the critical binding determinants of cellular retinoic acid binding protein II by mutagenesis and fluorescence binding assay

The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by providing a carboxylic acid dimer partner in the form of a Glu residue. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.     

Transporter-to-trap conversion: a disulfide bond formation in cellular retinoic acid binding protein I mutant triggered by retinoic acid binding irreversibly locks the ligand inside the protein

Transport proteins must bind their ligands reversibly to enable release at the point of delivery, while irreversible binding is usually associated with the extreme cases of ligand sequestration. Protein conformational dynamics is an important modulator of binding kinetics, as increased flexibility in the regions adjacent to the binding site may facilitate both association and dissociation processes. Ligand entry to, and exit from, the internal binding site of the cellular retinoic acid binding protein I (CRABP I) occurs via a flexible portal region, which functions as a dynamic aperture. We designed and expressed a CRABP I mutant (A35C/T57C), in which a small-scale conformational switch caused by the ligand binding event triggers formation of a disulfide bond in the portal region, thereby arresting structural fluctuations and effectively locking the ligand inside the binding cavity. At the same time, no formation of the disulfide bond is observed in the apo form of the mutant, and most characteristics of the mutant, including protein stability, are very similar to those of the wild-type protein in the absence of retinoic acid. The mutation does not alter the kinetics of retinoic acid binding to the protein, although the disulfide formation makes the binding effectively irreversible, as suggested by the absence of retinoic acid transfer from the holo form of the mutant to lipid vesicles in the absence of a reducing agent. Taken together, these data suggest that the disulfide bond formation in the portal region arrests large-scale structural fluctuations, which are required for retinoic acid release from the protein. The unique properties of the CRABP I mutant described in this work can be used to inspire and guide a design of nanodevices for multiple tasks ranging from sequestering small-molecule toxins in both tissue and circulation to nutrient deprivation of pathogens.     

Ligand-binding specificity of an invertebrate (Manduca sexta) putative cellular retinoic acid binding protein

Intracellular lipid-binding proteins (iLBPs) are small cytoplasmic proteins that specifically interact with hydrophobic ligands. Fatty acid-binding proteins (FABPs), cellular retinoic acid-binding proteins (CRABPs) and cellular retinol-binding proteins (CRBPs) belong to the iLBP family. A recently identified insect (Manduca sexta) iLBP has been reported to possibly represent an invertebrate CRABP mimicking the role of CRABPs in vertebrate organisms. The presence in this protein of the characteristic binding triad residues involved in the interaction with ligand carboxylate head groups, a feature pertaining to several FABPs and to CRABPs, and the close phylogenetic relationships with both groups of vertebrate heart-type FABPs and CRBPs/CRABPs, makes it difficult to assign it to either FABPs or CRABPs. However, its negligible interaction with retinoic acid and high affinity (K(d) values in the 10(-8) M range) for fatty acids have been established by means of direct and competitive binding assays. As shown by phylogenetic analysis, the M. sexta iLBP belongs to a wide group of invertebrate iLBPs, which, besides being closely related phylogenetically, share distinctive features, such as the conservation of chemically distinct residues in their amino acid sequences and the ability to bind fatty acids. Our results are in keeping with the lack of cellular retinoid-binding proteins in invertebrates and with their later appearance during the course of chordate evolution.     

斜纹夜蛾视黄酸结合蛋白(Slcrabp)基因的克隆、表达及功能

视黄酸结合蛋白(Cellular retinoic acid binding protein,CRABP)属于胞内脂质结合蛋白超基因家族,参与了许多生理活动,如细胞分化、组织重建和信号转导等,但其在昆虫中肠的作用尚不明确。研究从斜纹夜蛾Spodoptera litura中肠基因表达序列标签(EST)文库中克隆获得一个编码Slcrabp的全长c DNA,该c DNA由396个核苷酸组成,编码132个氨基酸。预测CRABP蛋白质的空间结构与脂肪酸结合蛋白非常相似,含一个由10个反平行的β折叠和2个α螺旋形成的配体结合中心结构域。Sl CRABP基因具有4个外显子,与脊椎动物crabp基因类似。RT-PCR检测表明,在转录水平上,Slcrabp在6龄幼虫中肠的各个时期均有较高表达。Western blot分析结果显示,Sl CRABP蛋白分布广泛,在中肠、脂肪体、精巢等组织上大量表达。在中肠,其表达峰值出现在预蛹期。利用荧光标记物质8-苯胺基-1-萘磺酸(1,8-ANS)分析了重组Sl CRABP蛋白与不同底物的亲和力,发现Sl CRABP与不饱和长链脂肪酸有较高结合活性,如花生四烯酸钠、亚麻酸、油酸钠和油酸,但与视黄酸、视黄醇的结合力却很弱或几乎不结合,暗示斜纹夜蛾Sl CRABP功能与同家族脂肪酸结合蛋白(FABP)性质相似。对6龄幼虫进行饥饿处理,结果显示经过经24 h和48 h饥饿处理后,虫体中肠Sl CRABP蛋白质的表达量有显著上升,暗示Sl CRABP可能参与了斜纹夜蛾体内脂类的转运过程。     

Calcium binding proteins and cellular regulation

An important feature of cellular regulation is the precise control of intracellular calcium levels. This is accomplished both by dynamic organelle release and sequestration of calcium and by specific calcium active transport mechanisms located in the plasma membrane. The actual calcium signal for mediation of a cellular response is carried out by specific intracellular proteins, the most widely studied examples are calmodulin and troponin C. The recent discovery of phospholipid protein kinase and calcimedins suggests receptor mediation via several independent proteins. The physiological importance of a particular protein as a calcium messenger rests on several features: 1) calcium binding is of the order of 1–10 μm, 2) the protein is known to be localized at the site of proposed action, 3) if translocation occurs upon activation, the time required is consistent with the time course of the physiologic response and 4) substrates or effectors at the next level of action when isolated can be demonstrated to have similar activation kinetics as $\text{in situ}$.