Protein and Peptide Letters - Volume 19, Issue 2, 2012

This Special Issue gathers eight timely review articles about the structure, function, and evolutionary relationships of the members of the alpha/beta-hydrolase fold superfamily of proteins. Most of the proteins in this superfamily display hydrolytic functions carried by a canonical triad of catalytic residues, which serve to catalyze the hydrolytic cleavage of a range of properly oriented substrates with various chemical characteristics. Yet a small set of the family members are devoid all or some of these catalytic residues, and several of these “non-hydrolase” proteins have been characterized at the structural and functional levels. However, these studies raised many questions, such as: To what extent can a protein perform two functions? Should the two functions be distinctive? Could they involve a single binding/recognition site or should the sites be distinctive? Can the two functions be performed simultaneously, or is each of them related to a specific cell or time environment dictating its turning on/off? Could a protein molecule switch from the one to the other function during evolution? The ESTHER database was designed 15 years ago to collect, annotate, organize and diffuse information related to the alpha/beta-hydrolase superfamily. The web server allows one to retrieve figures highlighting the need for more experimental data on structure and function of numerous proteins that are only related by sequence [1,2]. Indeed, among the thousands of proteins that share only limited sequence homology in the database, only 270 proteins belonging to 54 subfamilies have been crystallized, although this number increases steadily each year (figure 1). As well, in the human genome 121 genes have been shown to encode alpha/beta-hydrolase fold proteins, of which 20 apparently lack a functional catalytic site. A functional role has been described for only a few of the later, of which at least three (rbbp9, egasyn CIB) both display an enzymatic activity and participate in protein assemblies. Of the 31 human alpha/beta-hydrolases that have been crystallized, three (neuroligin, ndr2, dpp6) are not enzymes. Twenty-two members of the superfamily were shown to be responsible for genetic diseases when mutated, and modification of expression of many other members are suspected to be responsible for clinical symptoms in specific diseases. For a few of these, an anti-oncogene role has been reported (e.g., OVCA2). The characterization of other nonenzyme alpha/beta-hydrolases in insects, plants or bacteria can also shed light on evolution of this widely represented superfamily. This Special Issue encompasses eight chapters, corresponding to five reviews and three original articles. The opening chapter, a review by the guest editors P. Marchot and A. Chatonnet (France), provides readers with a panorama of the bestknown examples of protein-protein interactions in the most prominent families composing the superfamily, and presents some of the current knowledge about common or divergent modes of protein interactions [4]. In the second chapter, G. Manco and colleagues (Italy and China) review the various types of “promiscuity” encountered in the superfamily and provide examples of natural or directed evolution of enzymes in the hormone-sensitive lipase like subfamily [5]. In the third and fourth chapters, A. Vogel-Hopker and colleagues (Germany) and A. Halliday and S. Greenfield (Great Britain) review several of the pleiotropic functions of acetylcholinesterase, otherwise known as the enzyme that hydrolyzes the neurotransmitter acetylcholine to regulate functioning of cholinergic synapses, and a prototype in the COesterase family (Pfam PF00135) [6,7]. The fifth chapter, by A. De Jaco and colleagues (Italy and USA), reviews the natural mutations affecting the alpha/beta-hydrolase domain in an enzyme, in a synaptic cell adhesion protein and prototype of non-catalytic hydrolase in the family, and in a hormone receptor, and shows how these mutations impair processing and trafficking of the altered proteins along the secretory pathway [8]. In the sixth chapter, an original article by K. Hirano and colleagues (Japan) presents a comprehensive structural and functional analysis of the nuclear receptor to the gibberellin phytohormones, a non-hydrolase member of the hormone-sensitive lipase subfamily [9]. In the seventh chapter, an original article by C. Bahl and D. Madden (USA) reports the structure of an inactive mutant of Cif, a member of the epoxide hydrolase subfamily and virulence factor that reduces the functional localization of the CFTR channel in epithelial cells [10]. And the eighth chapter, an original article by S. Vorobiev and colleagues (USA), addresses the uncommon theme of assessing a hydrolytic function for a protein initially known for partnership interactions only [11]. Hence, we feel that this Special Issue, although not fully comprehensive, highlights the widely diverse aspects of promiscuity and moonlighting activities [12] performed by members of the alpha/beta-hydrolase fold superfamily of proteins. We thank all and each of the authors for their enthusiastic and efficient contribution to this Special Issue and patience towards the timetable and delays. We hope they approve the order in which the chapters are organized (a difficult choice) and are proud to share this literature contribution with us. At least two referees, selected among the authors or other colleagues, carefully reviewed each manuscript. We thank all the referees for the serious, constructive and efficient work.....

Genes coding for members of the alpha/beta hydrolase fold superfamily of proteins are present in all known genomes. Although there is no common and essential function performed by these proteins shared in all living organisms, this fold has been used for a number of diverse functions. The ancestry of both enzymatic and protein-protein interaction capability of this structural scaffold made it an important tinkering tool kit for protein function evolution. Recently, enzymes known since a long time have been found to have a second function in acting promiscuously on alternative substrates, or to be true moonlighting proteins acting also as transporters, receptors, chaperones… The reverse situation has been encountered for adhesion proteins shown to be enzymes. This review, while not exhaustive, surveys some of the best-known examples of multiple functions in alpha/beta hydrolase fold proteins.

The number of enzymes endowed with the capacity to catalyse other reactions than the main, physiological one, a feature that has been called promiscuity, is increasing at a fast pace. Promiscuity is a highly pervasive phenomenon that is present at each level of life complexity. For enzymes, promiscuity encompasses interesting aspects related to their physiological role, evolution and biotechnological applications. Herein, at first we will describe some general aspects of enzyme promiscuity and then we will report some examples from the α/β hydrolase superfamily of proteins, with particular emphasis to the hormone-sensitive lipase family.

Acetylcholinesterase (AChE) is a most remarkable protein, not only because it is one of the fastest enzymes in nature, but also since it appears in many molecular forms and is regulated by elaborate genetic networks. As revealed by sensitive histochemical procedures, AChE is expressed specifically in many tissues during development and in many mature organisms, as well as in healthy and diseased states. Therefore it is not surprising that there has been a long-standing search for additional, “non-classical” functions of cholinesterases (ChEs). In principle, AChE could either act nonenzymatically, e.g. exerting cell adhesive roles, or, alternatively, it could work within the frame of classic cholinergic systems, but in non-neural tissues. AChE might be considered a highly co-opting protein, since possibly it combines such various functions within one molecule. By presenting four different developmental cases, we here review i) the expression of ChEs in the neural tube and their close relation to cell proliferation and differentiation, ii) that AChE expression reflects a polycentric brain development, iii) the retina as a model for AChE functioning in neural network formation, and iv) nonneural ChEs in limb development and mature bones. Also, possible roles of AChE in neuritic growth and of cholinergic regulations in stem cells are briefly outlined.

Acetylcholinesterase (AChE), a member of the α/β-hydrolase fold superfamily of proteins, is a serine hydrolase responsible for the hydrolysis of the well studied neurotransmitter acetylcholine (ACh). However, it is becoming clear that AChE has a range of actions other than this ‘classical’ role. Non-classical AChE functions have been identified in apoptosis, stress-responses, neuritogenesis, and neurodegeneration. Furthermore, these non-classical roles are attributable not only to the native protein, which appears to act as a mediary binding protein under a number of circumstances, but also to peptides cleaved from the parent protein. Peptides cleaved from AChE can act as independent signalling molecules. Here we discuss the implications of non-hydrolytic functions of this multi-tasking protein.

The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold.

Gibberellins (GAs) are tetracyclic, diterpenoid plant hormones, essential for many developmental processes in higher plants. Plants perceive GA through a nuclear-localized GA receptor, GA INSENSITIVE DWARF1 (GID1). From sequence similarity, it is suggested that GID1 evolved from a hormone-sensitive lipase (HSL), and recent x-ray crystallography of the GA-GID1 complex has given insights into how GID1 recognizes GA. Analyses of the GA signaling pathway in several plant species further suggest that the GID1-mediated GA signaling pathway emerged in the vascular plant lineage and since then regulation of GA recognition specificity seems to have been fine tuned to strictly regulate the on-off GA signal.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the first epoxide ring-opening tyrosine in the active site. To test the functional equivalence of Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutant is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair.

Human retinoblastoma binding protein 9 (RBBP9) is an interacting partner of the retinoblastoma susceptibility protein (Rb). RBBP9 is a tumor-associated protein required for pancreatic neoplasia, affects cell cycle control, and is involved in the TGF-β signalling pathway. Sequence analysis suggests that RBBP9 belongs to the α/β hydrolase superfamily of enzymes. The serine hydrolase activity of RBBP9 is required for development of pancreatic carcinomas in part by inhibiting TGF-β antiproliferative signaling through suppressing Smad2/3 phosphorylation. The crystal structure of human RBBP9 confirms the α/β hydrolase fold, with a six-stranded parallel β-sheet flanked by α helixes. The structure of RBBP9 resembles that of the YdeN protein from Bacillus subtilis, which is suggested to have carboxylesterase activity. RBBP9 contains a Ser75-His165-Asp138 catalytic triad, situated in a prominent pocket on the surface of the protein. The side chains of the LxCxE sequence motif that is important for interaction with Rb is mostly buried in the structure. Structure- function studies of RBBP9 suggest possible routes for novel cancer drug discovery programs.

Rare earth elements (REEs), which include 17 elements in the periodic table, share chemical properties related to a similar external electronic configuration. REEs enriched fertilizers have been used in China since the 1980s. REEs could enter the cell and cell organelles, influence plant growth, and mainly be bound with the biological macromolecules. REE-binding proteins have been found in some plants. In addition, the chlorophyll activities and photosynthetic rate can be regulated by REEs. REEs could promote the protective function of cell membrane and enhance the plant resistance capability to stress produced by environmental factors, and affect the plant physiological mechanism by regulating the Ca2+ level in the plant cells. The focus of present review is to describe how REEs and REE-binding proteins participate in the physiological responses in plants.

Glucagon-like peptide-1 (GLP-1) was once considered as an ideal anti-diabetic candidate for its important role in maintaining glucose homeostasis through the regulation of islet hormone secretion, as well as hepatic and gastric function. However, the major therapeutic obstacle for using native GLP-1 as a therapeutic agent is its very short half-life primarily due to their degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). In this study, GLP-1 analogues with modifications in amino acid site 8, 22 and 23 were synthesized using solid phase peptide synthesis. Resistance of these analogues to DPP-IV cleavage was investigated in vitro by incubation of the peptides with DPP-IV or human plasma. Glucoregulating efficacy of the analogues was evaluated in normal Kunming mice using intraperitoneal glucose tolerance model. Glucose lowering effect of combination therapy (analogue plus Vildagliptin) has also been studied. In vitro studies showed that the modified analogues were much more stable than native GLP-1 (nearly 100% of the peptide keep intact after 4 h incubation). In vivo biological activity evaluation revealed that His8-EEE (the most potent GLP-1 analogues in this study) exhibited significantly improved glycemic control potency (approximately 4.1-fold over saline and 2.5-fold over GLP-1) and longer time of active duration (at least 5 h). Combination therapy also showed the trend of its superiority over mono-therapy. Modified analogues showed increased potency and biological half-time compared with the native GLP-1, which may help to understand the structure-activity relationship of GLP-1 analogues.

In an earlier study, we found PBP inhibited the progress of adjuvant-induced arthritis (AA). This study was aimed at evaluating the inhibitory effects of PBP in terms of NF-κB activation by using immunohistochemical and immunofluorescent technique in vitro and in vivo. IL-1β and TNF-α in serum were detected by method of ELISA. Immunofluorescent results showed that PBP inhibited NF-κB p65 translocation into nucleus. In vivo imaging showed that treatment with PBP decreased the enzyme labeling signal of NF-κB p65. Immunohistochemical staining revealed that PBP suppressed production of NF-κB p65 subunit in the joints and attenuated the productions of IL-1β and TNF-α in serum from AA. Moreover, NF-κB p65 nucleus translocation was prevented by simultaneous incubation with PBP and PGE2 was decreased by PBP through a feedback cycle. We report the first confirmation of the mimotope of PGE2 receptor EP4 modulatory action.

Hepcidin, a 25 amino acid peptide hormone containing a complex network of four disulfide bonds is the hormone regulator of iron homeostasis. Three bridges synthetic peptide analogs have been prepared following two synthetic strategies and two oxidation procedures: i) a microwave-assisted solid phase synthesis followed by air oxidation of the six free cysteines ii) a manual solid phase synthesis followed by stepwise deprotection and oxidation of cysteine pairs. All the peptides with different connectivities have been characterized by MALDI ToF spectrometry, and tested for their ability to degrade the cellular iron exporter, ferroportin. While linear peptides are inactive, the one-bridge and two-bridge peptides retaining protected cysteines by bulky substituents are active. Similarly, the three-bridge peptides are active irrespective of their disulfide connectivities.

Shiga toxins are one of the very potent agents for causing dysentery, diarrhoea and haemolytic uremic syndrome with very low LD50. For better understanding of their biology, detection and neutralization, the components of toxins are needed to be expressed and purified in bulk amounts. However, following traditional expression procedures, this task is very tedious as the yield of the toxin is very low. In this manuscript, we have described the optimization of media for enhanced production of recombinant Shiga toxin B (rStx-B) chain protein in Escherichia coli. This protein is known to have neutralization ability against shiga toxins. Furthermore, fed-batch cultivation process in E. coli was also developed in the optimized medium. Expression was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG). The purification of protein involved Ni-NTA affinity chromatography under native conditions followed by gel filtration chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell weight and purified protein of about 19.41 g/l (dry cell weight, 11.38 g/l) and 30 mg/l of culture, respectively. The purity of the recombinant StxB protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Reactivity of this protein was determined by Western blotting as well as indirect ELISA using specific antibodies. These results establish the application of this protein for diagnosis of shiga toxin infection or for neutralizing the toxicity.

Icarapin is a bee venom protein found to induce IgE-mediated allergic reaction. In this study, icarapin of Asian honey bee was cloned and sequenced. By in silico screening, S198 was found to be the potential antigenic site. This site was changed to cysteine and coupled with PEG5K. Compared to the wild type icarapin and the S198C variant, PEGylated S198C variant induced lower level of IgG and IgE antibodies in mice, showing that it is indeed located in an antigenic site. Our work may be generalized to other proteins for the discovery of antigenic sites and the reduction of antigenicity

As an alternative to X-ray crystallography, nuclear magnetic resonance (NMR) has also emerged as the method of choice for studying both protein structure and dynamics in solution. However, little work using computational models such as Gaussian network model (GNM) and machine learning approaches has focused on NMR-derived proteins to predict the residue flexibility, which is represented by the root mean square deviation (RMSD) with respect to the average structure. We provide a large-scale comparison of computational models, including GNM, parameter-free GNM and several linear regression models using local solvent exposures as inputs, based on a dataset of 1609 protein chains whose structures were resolved by NMR. The result again confirmed that the correlation of GNM outputs with raw RMSD values was better than that using B-factors of X-ray data. Nevertheless, it was also concluded that the parameter-free GNM and the solvent exposure based linear regression models performed worse than GNM when predicting RMSD, contrary to results using X-ray data. The discrepancy of residue flexibility prediction between NMR and X-ray data is likely attributable to a combination of their physical and methodological differences.