Novel fluorescent phosphonic acid esters for discrimination of lipases and esterases

Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)selective modification of synthetic substrates. Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations. The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis. The selectivities of the inhibitors were determined by using different (phospho)lipase, esterase and cholesterol esterase preparations. The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor. We identified inhibitors with very low specificity, and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases. A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.     

A lateral flow protein microarray for rapid and sensitive antibody assays

Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.     

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.     

Polysaccharide microarrays for polysaccharide-platelet-derived-growth-factor interaction studies

Polysaccharide microarrays have great potential for the high-throughput analysis of polysaccharide-protein interactions. Here we demonstrate that a polysaccharide microarray prepared by printing a library of dextran polymers derivatized by methylcarboxylate, benzylamide, and sulfate groups (DMCBSu compounds) on to glass slides permitted the rapid identification of a set of compounds able to interact with the platelet-derived growth factor BB (PDGF-BB) isoform, a growth factor involved in wound healing. Microarray interaction results were compared to the capacity of DMCBSu compounds to potentiate the in vitro PDGF-BB-induced proliferation of human dermal fibroblasts.     

Protein Microarray Copying: Easy on-Demand Protein Microarray Generation Compatible with Fluorescence and Label-Free Real-Time Analysis

Protein microarrays are essential to understand complex protein interaction networks. Their production, however, is a challenge and renders this technology unattractive for many laboratories. Recent developments in cell-free protein microarray generation offer new opportunities, but are still expensive and cumbersome in practice. Herein, we describe a cost-effective and user-friendly method for the cell-free production of protein microarrays. From a polydimethylsiloxane (PDMS) flow cell containing an expressible DNA microarray, proteins of interest are synthesised by cell-free expression and then immobilised on a capture surface. The resulting protein microarray can be regarded as a “copy” of the DNA microarray. 2 His₆- and Halo-tagged fluorescent reference proteins were used to demonstrate the functionality of nickel nitrilotriacetic acid (Ni-NTA) and Halo-bind surfaces in this copy system. The described process can be repeated several times on the same DNA microarray. The identity and functionality of the proteins were proven during the copy process by their fluorescence and on the surface through a fluorescent immune assay. Also, single-colour reflectometry (SCORE) was applied to show that, on such copied arrays, real-time binding kinetic measurements were possible.     

On the hyperthermostability of lipolytic enzymes from Thermus aquaticus YT‐1: exploring their application to polymer degradation

BACKGROUND: The investigation of enzymes from extremophilic microorganisms has generated great scientific interest in recent decades owing to their ability to operate in harsh conditions. Since lipolytic enzymes are one of the three groups most commercially demanded, the study of novel thermostable lipolytic enzymes is of much interest. RESULTS: Lipolytic enzymes produced by Thermus aquaticus YT1 have been investigated. Very interesting properties against thermal inactivation, with a half‐life time of 1 h at 95 °C were found. The use of compounds such as polyols is a strategy used to further improve the thermostability of these enzymes, and the modelling of the deactivation process with a series‐type equation, allows one to quantitatively ascertain the free energy of deactivation. Finally, the application to dibutyl adipate hydrolysis was investigated, as an indicator of the potential usefulness of the enzymes in polymer biodegradation. CONCLUSION: The crude lipolytic enzymes possess outstanding properties of thermostability (high lipolytic activity after 12 days at 80 °C, and a half‐life time of 1 h at 95 °C), and they can be significantly improved by adding polyols (addition of glycerol entailed activity losses of just 3% after 1 h at 95 °C). These enzymes are able to catalyze ester cleavage, showing a zero‐order kinetics. Copyright © 2011 Society of Chemical Industry     

Protein microarrays and biomarkers of infectious disease

Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers.     

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin‐3 Inhibitor

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent‐field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3′‐mono‐ and 2,3′‐diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin‐3 for 2′‐arylamido derivatives over ureas, thioureas, and amines and that of galectin‐7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin‐3 versus galectin‐1 and galectin‐7.     

A Quantitative Systems Approach to Define Novel Effects of Tumour p53 Mutations on Binding Oncoprotein MDM2

Understanding transient protein interactions biochemically at the proteome scale remains a long-standing challenge. Current tools developed to study protein interactions in high-throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self-assembling protein microarrays produced by freshly translating full-length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label-free platform that measures many protein interactions in real-time allowing the determination of the KDs and rate constants. The developed novel NAPPA-SPRi technique showed excellent ability to study protein-protein interactions of clinical mutants of p53 with its regulator MDM2. Furthermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin-3, currently in clinical practice, which usually disrupts the p53-MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg-273-Cys, Arg-273-His, Arg-248-Glu, Arg-280-Lys), on the structural domain (His-179-Tyr, Cys-176-Phe), on hydrophobic moieties in the DNA binding domain (Arg-280-Thr, Pro-151-Ser, Cys-176-Phe) and hot spot mutants (Gly-245-Cys, Arg-273-Leu, Arg-248-Glu, Arg-248-Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.     

Cell-Permeable Ubiquitin and Histone Tools for Studying Post-translational Modifications

Protein post-translational modifications (PTMs) regulate nearly all biological processes in eukaryotic cells, and synthetic PTM protein tools are widely used to detect the activity of the related enzymes and identify the interacting proteins in cell lysates. Recently, the study of these enzymes and the interacting proteome has been accomplished in live cells using cell-permeable PTM protein tools. In this concept, we will introduce cell penetrating techniques, the syntheses of cell-permeable PTM protein tools, and offer some future perspective.     

Lipochitin Oligosaccharides Immobilized through Oximes in Glycan Microarrays Bind LysM Proteins

Glycan microarrays have emerged as novel tools to study carbohydrate–protein interactions. Here we describe the preparation of a covalent microarray with lipochitin oligosaccharides and its use in studying proteins containing LysM domains. The glycan microarray was assembled from glycoconjugates that were synthesized by using recently developed bifunctional chemoselective aminooxy reagents without the need for transient carbohydrate protecting groups. We describe for the first time the preparation of a covalent microarray with lipochitin oligosaccharides and its use for studying proteins containing LysM domains. Lipochitin oligosaccharides (also referred to as Nod factors) were isolated from bacterial strains or chemoenzymatically synthesized. The glycan microarray also included peptidoglycan‐related compounds, as well as chitin oligosaccharides of different lengths. In total, 30 ligands were treated with the aminooxy linker molecule. The identity of the glycoconjugates was verified by mass spectrometry, and they were then immobilized on the array. The presence of the glycoconjugates on the array surface was confirmed by use of lectins and human sera (IgG binding). The functionality of our array was tested with a bacterial LysM domain‐containing protein, autolysin p60, which is known to act on the bacterial cell wall peptidoglycan. P60 showed specific binding to Nod factors and to chitin oligosaccharides. Increasing affinity was observed with increasing chitin oligomer length.     

Chloroplastic glycolipids fuel aldehyde biosynthesis in the marine diatom Thalassiosira rotula

Enzymatic preparations and specialized analytical tools have shown that chloroplast-derived glycolipids are the main substrates for the biosynthetic pathway that produces antiproliferative polyunsaturated aldehydes in broken cells of the marine diatom Thalassiosira rotula. This process, which is associated with the formation of free fatty acids and lyso compounds from polar lipids but not triglycerides, is largely dependent on glycolipid hydrolytic activity, rather than phospholipase A(2) as previously suggested. Preliminary characterization of lipolytic enzymes has revealed protein bands of 40-45 kDa. Under native conditions these proteins seem to be associated with soluble aggregates that have an apparent molecular weight of approximately 200 kDa. The biochemical process, which is similar to that described in the algal-bloom forming diatom Skeletonema costatum, suggests a mechanism based on decompartmentalization and mixing of preexisting enzymes and substrates.     

Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases

Functional screenings were conducted on two metagenomic libraries from hot springs in order to find novel thermozymes with potential biotechnological applications. These included enzymes acting on plant cell walls such as endoglucanases and exoglucanases, β-glucosidases, xylanases, and β-xylosidases, and broad application enzymes such as proteases and lipolytic hydrolases. Of all the enzymes found by this bioprospection, we selected a novel lipolytic enzyme for further characterization. The protein was found to belong to the SGNH/GDSL family of hydrolases. It was purified and its biochemical parameters determined. We found that the enzyme was most active at 60 °C and pH 9 using pNP-laurate as substrate and was highly thermostable. It also showed preference for short-chained substrates and activation with temperature and with certain detergents such as Tween 80. Proteins of this family of hydrolases are relevant for their broad substrate specificity, that coupled with this protein’s high temperature optima, broad pH range, and thermostability further highlights its biotechnological potential.     

Development of Selective Bisubstrate‐Based Inhibitors Against Protein Kinase C (PKC) Isozymes By Using Dynamic Peptide Microarrays

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP‐binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate‐based inhibitors that target the more selective peptide‐binding site in addition to the ATP‐binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate‐based leads. Dynamic peptide microarrays were used to find peptide‐binding site inhibitors. These were linked with chemoselective click chemistry to an ATP‐binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate‐based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCθ amongst three isozymes.     

A Comparison of the Levels of Oil,Carotenoids, and Lipolytic Enzyme Activities in Modern Lines and Hybrids of Grain Sorghum

Carotenoids are potentially valuable components in grain sorghum and there is a need to better understand their concentration, composition, and value. Thirteen modern commercial grain sorghum hybrids and five sorghum lines were extracted and analyzed and the levels of oil and carotenoids were compared. The same samples were also evaluated for lipolytic enzyme activity. The oil content in all eighteen samples ranged from 3.21 to 4.29 wt%. Lutein and zeaxanthin were the predominant carotenoids and the levels of total carotenoids ranged from 3.82 to 19.5 ppm in the oil, which was much lower than the levels of total carotenoids in two yellow corn samples (70.8 and 103 ppm). Lipolytic enzyme activity was estimated by storing milled kernel samples for 2 weeks at 25 °C and measuring the levels of total free fatty acids. After 2 weeks, the levels of free fatty acids in the oil of the eighteen sorghum samples ranged from 11.49 and 52.17 wt%, compared to 10.29 and 17.54 wt% in oil from the two corn samples. This new data will be useful for persons using grain sorghum for food, biofuels and other industrial applications. Examples of sorghum genotypes with high and low levels of carotenoids and high and low levels of lipolytic enzymes were both identified.     

Enzyme Therapy: Current Challenges and Future Perspectives

In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.     

Self-assembled small-molecule microarrays for protease screening and profiling

Small-molecule microarrays are attractive for chemical biology as they permit the analysis of hundreds to thousands of interactions in a highly miniaturized format. Methods to prepare small-molecule microarrays from combinatorial libraries by a self-assembly process based on the sequence-specific hybridization of peptide nucleic acid (PNA) encoded libraries to oligonucleotide arrays are presented. A systematic study of the dynamic range for multiple detection agents, including direct fluorescence of attached fluorescein and cyanine-3 dyes, antibody-mediated fluorescence amplification, and biotin-gold nanoparticle detection, demonstrated that individual PNA-encoded probes can be detected to concentrations of 10 pM on the oligonucleotide microarrays. Furthermore, a new method for parallel processing of biological samples by using gel-based separation of probes is presented. The methods presented in this report are exemplified through profiling two closely related cysteine proteases, cathepsin K and cathepsin F, across a 625-member PNA-encoded tetrapeptide acrylate library. A series of the specific cathepsin K and F inhibitors identified from the library were kinetically characterized and shown to correlate with the observed microarray profile, thus validating the described methods. Importantly, it was shown that this method could be used to obtain orthogonal inhibitors that displayed greater than tenfold selectivity for these closely related cathepsins.     

Lipolytic Enzyme Production by Immobilized Rhizopus oryzae

In this paper, a suitable technique as well as an adequate material for the immobilization of the fungus Rhizopus oryzae were investigated. This organism has been shown to have potential in the field of food aroma due to the production of extracellular lipolytic enzymes. However, an efficient production system at bioreactor scale for its application to the flavor compounds production is still needed. Among the supports studied, alginate beads were the best carrier materials, leading to the highest lipolytic activities of up to 400 U/L after 3 days of cultivation. Repeated batch cultures were carried out to improve cell concentration and lipolytic activity. The gel beads produced lipolytic enzyme under optimized conditions for consecutive batch fermentations without marked activity loss and deformation attained a maximum level of 715 U/L after three batches. The production of lipolytic enzyme by immobilized Rhizopus oryzae in a 2‐litre airlift bioreactor with the optimized conditions was evaluated. Lipolytic activities of 487 U/L were attained, operating in successive batches without operational problems and the bioparticles (the fungus grows in alginate beads) maintained their shape throughout fermentation.     

Site‐Selective Protein Immobilization through 2‐Cyanobenzothiazole‐Cysteine Condensation

We described a rapid site‐selective protein immobilization strategy on glass slides and magnetic nanoparticles, at either the N or C terminus, by a 2‐cyanobenzothiazole (CBT)‐cysteine (Cys) condensation reaction. A terminal cysteine was generated at either terminus of a target protein by a combination of expressed protein ligation (EPL) and tobacco etch virus protease (TEVp) digestion, and was reacted with the CBT‐solid support to immobilize the protein. According to microarray analysis, we found that glutathione S‐transferase immobilized at the N terminus allowed higher substrate binding than for immobilization at the C terminus, whereas there were no differences in the activities of N‐ and C‐terminally immobilized maltose‐binding proteins. Moreover, immobilization of TEVp at the N terminus preserved higher activity than immobilization at the C terminus. The success of utilizing CBT‐Cys condensation and the ease of constructing a terminal cysteine using EPL and TEVp digestion demonstrate that this method is feasible for site‐selective protein immobilization on glass slides and nanoparticles. The orientation of a protein is crucial for its activity after immobilization, and this strategy provides a simple means to evaluate the preferred protein immobilization orientation on solid supports in the absence of clear structural information.     

Lipolytic activity of California-laurel (Umbellularia californica) seeds

The lipid content of dormant mature seeds of the California-laurel (Umbellularia califonnica) was 74% (mass basis) and decreased upon germination, reaching 43% 5 wk after germination. Dormant seeds contained only barely detectable lipase activity. Lipase activity rose upon germination, peaking 2 to 3 wk after the onset of visible germination. The combined addition of three detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (1 mM), taurocholic acid (1 mM), and Tween-20 (0.05%, vol/vol) during homogenization increased the lipolytic activity of total seedling homogenates by about 60%. Following centrifugation of homogenates from seedlings of various ages, ca. 80% of the recovered lipolytic activity was located in the fat-free supernatant, with the remainder in the floating fat pad. The crude seed lipase did not show hydrolytic specificity for glycerol esters of lauric acid (the predominant fatty acid of the seed triglycerol): comparable lipolytic activities were seen toward olive oil, trilaurin, tripalmitin, and tristearin. Maximal lipolytic activity occurred at pH 8.5. This activity was stable over the pH range 6 to 9, and unstable at >40°C in a fashion that suggested the presence of multiple enzymes with different substrate specificities. Two lipolytic species, one of which showed some selectivity toward lauric acid esters, were partially separated from one another by ion-exchange chromatography.