Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases

An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.     

Characterization of two aldo–keto reductases from Gluconobacter oxydans 621H capable of regio- and stereoselective α-ketocarbonyl reduction

Two cytosolic NADPH-dependent carbonyl reductases from Gluconobacter oxydans 621H, Gox0644 and Gox1615, were heterologously produced in Escherichia coli. The recombinant proteins were purified to homogeneity and characterized. Gox0644 and Gox1615 were dimers with native molecular masses of 66.1 and 74.5 kDa, respectively. The enzymes displayed broad substrate specificities and reduced α-ketocarbonyls at the keto moiety most proximal to the terminus of the alkyl chain to produce alpha-hydroxy carbonyls, as demonstrated by NMR. With respect to stereoselectivity, protein Gox0644 specifically reduced 2,3-pentanedione to 2R-hydroxy-pentane-3-one, whereas Gox1615 produced 2S-hydroxy-pentane-3-one. Both enzymes also reduced 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenylpropane-1-one, which is a key intermediate in the production of numerous pharmaceuticals, such as antifungal azoles and antidepressants. Gox0644 displayed highest activities with 2,3-diones, α-ketoaldehydes, α-keto esters, and 2,5-diketogluconate. Gox1615 was less active with these substrates, but displayed a broader substrate spectrum reducing a variety of α-diketones and aldehydes.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

Expressed protein ligation to study protein interactions: semi-synthesis of the G-protein alpha subunit

Interactions between G proteins and GPCRs are fundamental for transmitting signals for a multitude of physiologic responses. Little is known regarding the protein-protein interface between the G protein and the receptor, much less the mechanisms for receptor activation of G proteins. Here, we will describe how expressed protein ligation will aid in the study of protein-protein interactions between semi-synthetic G alpha subunits and GPCRs.     

Identification and functional characterization of four novel aldo/keto reductases in <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 by integrating wet lab with in silico approaches

Aldo/keto reductases (AKRs) constitute a multitasking protein family that catalyzes diverse metabolic transformations including detoxification of stress generated reactive aldehydes. Yet this important protein family is poorly understood particularly in cyanobacteria, the ecologically most diverse and significant group of micro-organisms. Present study is an attempt to characterize all putative AKRs of Anabaena sp. PCC 7120. In silico analysis, it revealed the presence of at least four putative AKRs in Anabaena PCC7120 genome. All four proteins share less than 40% sequence identity with each other and also with the identified members of AKR superfamily and hence deserve to be assigned in new families. Dissimilarity in sequences is also reflected through their substrate specificity. While reduction of trans-2-nonenal, a LPO-derived reactive aldehyde was common across the four proteins, these proteins were found to be activated during heat, salt, Cd, As, and butachlor treatments, and their ectopic expression in Escherichia coli conferred tolerance to the above abiotic stresses. These findings affirm the role of AKRs in providing a broad tolerance to environmental stresses conceivably by detoxifying the stress-generated reactive aldehydes.     

Expressed protein ligation: a new tool for the biosynthesis of cyclic polypeptides

The present paper reviews the use of expressed protein ligation for the biosynthesis of backbone cyclized polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes.     

Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein

Advances in expressed protein ligation (EPL) methods that permit specific introduction of unique modifications into proteins have facilitated protein engineering, structure-function and protein interaction studies. An EPL-generated hybrid exchangeable apolipoprotein has been constructed from recombinant fragments of apolipoprotein E (apoE) and apolipophorin III (apoLp-III). A recombinant fusion protein comprised of human apoE N-terminal residues 1-111, a modified Saccharomyces cerevisiae intein and a chitin binding domain was subjected to 2-mercaptoethanesulfonic acid (MESNA) induced cleavage to generate apoE(1-111)-MESNA. A second fusion protein was comprised of a bacterial pelB leader peptide fused to a variant form of Galleria mellonella apoLp-III residues 1-91. The N-terminal pelB leader sequence directed the newly synthesized fusion protein to the Escherichia coli perisplamic space where endogenous leader peptidase cleavage generated the desired N-terminal cysteine-containing protein fragment. The resulting apoLp-III fragment, which contained no sequence tags or tails, escaped the bacteria and accumulated in the culture medium. When cultured in M9 minimal medium, Asp1Cys apoLp-III(1-91) was produced in high yield and was the sole major protein in the culture supernatant. Ligation reactions with apoE(1-111)-MESNA yielded an engineered hybrid apolipoprotein. The results document the utility of the pelB fusion protein system for generating active N-terminal cysteine containing proteins for EPL applications.     

Chemical ligation to obtain proteins comprising helices with individual amino acid sequences

Development of the strategies for assembling multiple kinds of peptide segments would give new possibilities for the de novo design of functional proteins. We will introduce our approach for the selective assembly of helical peptide segments on a peptide template to give four-helix-bundle proteins comprising individual helices.     

Overproduction of an Arabidopsis aldo–keto reductase increases barley tolerance to oxidative and cadmium stress by an in vivo reactive aldehyde detoxification

In this work, we expressed an Arabidopsis thaliana-coded protein (AKR4C9) in transgenic barley to study its enzymatic activity and to enhance the reactive aldehyde neutralizing capacity (part of the oxidative stress tolerance) of transgenic plants. Total leaf protein was extracted from transgenic plants expressing either C or N-terminally His-tagged aldo–keto reductase (AKR) enzyme and purified by affinity chromatography. The Arabidopsis-coded enzyme showed moderate activity against the synthetic reactive aldehyde, glutaraldehyde, and low but detectable enzyme activity against fructose with a low Michaelis–Menten constant (K_m value). Activity of the C and the N-terminally His-tagged AKRs were found to be in the same range. Glutaraldehyde was also tested in vivo by spraying onto the leaves of the plants. The reactive aldehyde tolerance of both wild type and transgenic plants, as well as the general physiological effects of this reactive aldehyde treatment were evaluated. The growth rate was found to decrease in all (both wild type and transgenic) plants. The high AKR-expressing transgenic plants showed a lower respiratory rate, and they also showed higher fresh weight, higher chlorophyll content and photosynthetic activity, indicating a higher reactive aldehyde tolerance. Cadmium (Cd) treatment was also performed to validate this result. Cd caused strong lipid peroxidation; however, the Arabidopsis enzyme lowered the reactive aldehyde content as expected. This is the first report in which kinetic parameters of the fructose reduction by the stress inducible plant AKR enzyme are presented. Furthermore, data on the effects of a reactive aldehyde treatment on intact plants are also provided.     

Phosphorylation of eIF4E attenuates its interaction with mRNA 5' cap analogs by electrostatic repulsion: intein-mediated protein ligation strategy to obtain phosphorylated protein

Phosphorylation of the eukaryotic initiation factor eIF4E in response to mitogenic stimuli and cytokines is implicated in the regulation of the initiation step of translation. It still remains unclear how the phosphorylation of eIF4E regulates the translation. To address this problem, we applied a unique technique in protein engineering, intein-mediated protein ligation, to synthesize eIF4E, which is selectively phosphorylated at Ser 209. Using selectively chosen synthetic cap analogs, we compared quantitatively the cap affinity for phosphorylated and unphosphorylated eIF4E by a fluorometric time-synchronized titration method. A 1.5- to 4.5-fold reduction of the cap affinity for phosphorylated eIF4E was observed, depending on the negative charge of the 5'-to-5' phosphate chains as well as the presence of a longer tetraribonucleotide strand. Possible implications for understanding the regulation of eIF4E functioning, cap complex formation, and stability, are discussed.     

Tolerance to 1-pentene-3-ol and to 1-pentene-3-one in relation to alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities inDrosophila melanogaster

The detoxification of 1-pentene-3-ol (pentenol) and 1-pentene-3-one (pentenone) by Drosophila melanogaster adult flies has been studied in two homozygous lines for the AdhF and AdhS alleles (LRC lines), in their respective lines selected for tolerance to ethanol (LRSe lines) and in a homozygous strain for the Adhn4 null allele. For each line, the genotype and sex LDs50 of both compounds were estimated. Then, in order to explain the differences in LD50, both alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities were assayed. In addition, the effects of pentenone on AKR activity were also studied. Our results show that ADH-positive flies exhibit a much higher sensitivity to pentenol than ADH-null flies. However, both ADH-positive and ADH-null flies show a similar tolerance to pentenone. Our results show that flies selected for improving tolerance to ethanol also have increased tolerance to pentenol (FF and SS flies) and pentenone (SS flies). However, this improved ability to tolerate pentenol and/or pentenone cannot be explained by changes in ADH or AKR activities. On the other hand, we have observed a beneficial effect of pentenol, but not of pentenone, in n4 flies. We also show that AKR activity is not modified by the administration of pentenone. These results suggest that, in the absence of ADH activity, pentenol may be transformed into a compound that is less toxic than pentenone and that pentenone itself might also be transformed into a less toxic compound.     

Overexpression of <Emphasis Type="Italic">GmAKR1</Emphasis>, a stress-induced aldo/keto reductase from soybean,retards nodule development

Development of symbiotic root nodules in legumes involves the induction and repression of numerous genes in conjunction with changes in the level of phytohormones. We have isolated several genes that exhibit differential expression patterns during the development of soybean nodules. One of such genes, which were repressed in mature nodules, was identified as a putative aldo/keto reductase and thus named Glycine max aldo/keto reductase 1 (GmAKR1). GmAKR1 appears to be a close relative of a yeast aldo/keto reductase YakC whose in vivo substrate has not been identified yet. The expression of GmAKR1 in soybean showed a root-specific expression pattern and inducibility by a synthetic auxin analogue 2,4-D, which appeared to be corroborated by presence of the root-specific element and the stress-response element in the promoter region. In addition, constitutive overexpression of GmAKR1 in transgenic soybean hairy roots inhibited nodule development, which suggests that it plays a negative role in the regulation of nodule development. One of the Arabidopsis orthologues of GmAKR1 is the ARF-GAP domain 2 protein, which is a potential negative regulator of vesicle trafficking; therefore GmAKR1 may have a similar function in the roots and nodules of legume plants. These authors contributed equally to this work.     

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.     

Recent advances in the application of expressed protein ligation to protein engineering

Expressed protein ligation is a technique for joining recombinantly expressed proteins to polypeptides containing biophysical probes, post-translational modifications or unnatural amino acids. Recent advances have expanded the scope of expressed protein ligation and have allowed the approach to be applied to the study of basic biological questions.     

A rapid and efficient way to obtain modified chemokines for functional and biophysical studies

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts.     

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior.     

Porcine odorant-binding protein selectively binds to a human olfactory receptor

Odorant-binding proteins (OBPs) represent a highly abundant class of proteins secreted in the nasal mucus by the olfactory neuroepithelium. These proteins display binding affinity for a variety of odorant molecules, thereby assuming the role of carrier during olfactory perception. However, no specific interaction between OBP and olfactory receptors (ORs) has yet been shown and early events in olfaction remain so far poorly understood at a molecular level. Two human ORs, OR 17-209 and OR 17-210, were fused to a Green Fluorescent Protein and stably expressed in COS-7 cell lines. Interaction with OBP was investigated using a highly purified radioiodinated porcine OBP (pOBP) preparation, devoid of any ligand in its binding cavity. No specific binding of the pOBP tracer could be detected with OR 17-209. In contrast, OR 17-210 exhibited specific saturable binding (K(d) = 9.48 nM) corresponding to the presence of a single class of high-affinity binding sites (B(max) = 65.8 fmol/mg prot). Association and dissociation kinetics further confirmed high-affinity interaction between pOBP and OR 17-210. Autoradiographic studies of labeled pOBP to newborn mouse slices revealed the presence of multiple specific binding sites located mainly in olfactory tissue but also in several other peripheral tissues. Our data thus demonstrate a high-affinity interaction between OBP and OR, indicating that under physiological conditions, ORs may be specifically associated with an OBP partner in the absence of odorant. This provides further evidence of a novel role for OBP in the mechanism of olfactory perception.