Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes

The field of biochemistry is currently faced with the enormous challenge of assigning functional significance to more than thirty thousand predicted protein products encoded by the human genome. In order to accomplish this daunting task, methods will be required that facilitate the global analysis of proteins in complex biological systems. Recently, methods have been described for simultaneously monitoring the activity of multiple enzymes in crude proteomes based on their reactivity with tagged chemical probes. These activity based probes (ABPs) have used either radiochemical or biotin/avidin-based detection methods to allow consolidated visualization of numerous enzyme activities. Here we report the synthesis and evaluation of fluorescent activity based probes for the serine hydrolase super-family of enzymes. The fluorescent methods detailed herein provide superior throughput, sensitivity, and quantitative accuracy when compared to previously described ABPs, and provide a straight-forward platform for high-throughput proteome analysis.     

Trifunctional chemical probes for the consolidated detection and identification of enzyme activities from complex proteomes

Chemical probes that covalently modify the active sites of enzymes in complex proteomes are useful tools for identifying enzyme activities associated with discrete (patho) physiological states. Researchers in proteomics typically use two types of activity-based probes to fulfill complementary objectives: fluorescent probes for rapid and sensitive target detection and biotinylated probes for target purification and identification. Accordingly we hypothesized that a strategy in which the target detection and target isolation steps of activity-based proteomic experiments were merged might accelerate the characterization of differentially expressed protein activities. Here we report the synthesis and application of trifunctional chemical proteomic probes in which elements for both target detection (e.g. rhodamine) and isolation (e.g. biotin) are appended to a sulfonate ester reactive group, permitting the consolidated visualization and affinity purification of labeled proteins by a combination of in-gel fluorescence and avidin chromatography procedures. A trifunctional phenyl sulfonate probe was used to identify several technically challenging protein targets, including the integral membrane enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-isomerase and the cofactor-dependent enzymes platelet-type phosphofructokinase and type II tissue transglutaminase. The latter two enzyme activities were significantly up-regulated in the invasive estrogen receptor-negative (ER(-)) human breast cancer cell line MDA-MB-231 relative to the non-invasive ER(+) breast cancer lines MCF7 and T-47D. Collectively these studies demonstrate that chemical proteomic probes incorporating elements for both target detection and target isolation fortify the important link between the visualization of differentially expressed enzyme activities and their subsequent molecular identification, thereby augmenting the information content achieved in activity-based profiling experiments.     

Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients

Diabetes can lead to serious microvascular complications like proliferative diabetic retinopathy (PDR), which is the leading cause of blindness in adults. The proteomic changes that occur during PDR cannot be measured in the human retina for ethical reasons, but could be reflected by proteomic changes in vitreous humor. Thus, we considered that comparisons between the proteome profiles of the vitreous humors of PDR and nondiabetic controls could lead to the discovery of novel pathogenic proteins and clinical biomarkers. In this study, the authors used several proteomic methods to comprehensively examine vitreous humor proteomes of PDR patients and nondiabetic controls. These methods included immunoaffinity subtraction (IS)/2-DE/MALDI-MS, nano-LC-MALDI-MS/MS, and nano-LC-ESI-MS/MS. The identified proteins were subjected to the Trans-Proteomic Pipeline validation process. Resultantly, 531 proteins were identified, i.e., 415 and 346 proteins were identified in PDR and nondiabetic control vitreous humor samples, respectively, and of these 531 proteins, 240 were identified for the first time in this study. The PDR vitreous proteome was also found to contain many proteins possibly involved in the pathogenesis of PDR. The proteins described provide the most comprehensive proteome listing in the vitreous humor samples of PDR and nondiabetic control patients.     

Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors

Serine hydrolases are a diverse enzyme class representing ～1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.     

Activity-based proteomics: enzymatic activity profiling in complex proteomes

Summary. In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine proteases, tyrosine phosphatases, glycosidases, and others. These probes are usually detected by their fluorescent, radioactive or affinity tags and their protein targets are analyzed using established proteomics techniques. Recent developments, such as the design of probes for in vivo analysis of proteomes, as well as microarray technologies for higher throughput screenings of protein specificity and the application of activity-based probes for drug screening are highlighted. We focus on biological applications of activity-based probes for target and inhibitor discovery and discuss challenges for future development of this field.     

Antibody microarray analysis of directly labelled complex proteomes

In recent years, the antibody microarray technology has made significant progress, going from proof-of-concept designs to established high-performing technology platforms capable of targeting non-fractionated complex proteomes. In these cross-disciplinary efforts, a particular focus has lately been placed on two key technological issues: the sample and data handling. To this end, robust protocols have been designed for direct labelling of whole proteomes compatible with a sensitive fluorescent-based sensing. Tagging of the proteins with biotin in a single-colour approach has, in many cases, proven to be the preferred approach. Furthermore, based on modified approaches, adopted from the DNA microarray field, the first bioinformatic standards for performing the antibody microarray data analysis have emerged, though general standard operating procedure(s) remains to be implemented.     

Comparisons of Peptide hydrolase activities in cereals

Carboxypeptidase activity (hydrolysis of N-carbobenzoxy-l-phenylalanyl-l-alanine) is high in a number of temperate zone cereals, originating in Asia Minor (wheat, barley, oats, wild oats, rye, triticale) compared to other cereals originating in central America or Asia (maize, sorghum, rice). However, endopeptidase activity (hydrolysis of azocasein or hemoglobin) is relatively much higher in the latter group. Comparison of trichloroacetic acid (TCA)-soluble products derived from the hydrolysis of hemoglobin showed that carboxyterminal amino acids (histidine, arginine, and tyrosine), are released when extracts from wheat and barley endosperms are used. With extracts from corn endosperms, much more TCA-soluble ultraviolet- absorbing material is released, but very little is released as free amino acids within the first 2 hours and the expected C-terminal amino acids of hemoglobin are not detected in significant amounts. These results suggest that the method of hydrolysis of the storage proteins may be significantly different in these two classes of cereals.     

Determination of the active-site serine of 6-aminohexanoate-dimer hydrolase

Diisopropylfluorophosphate, an inhibitor of serine proteinase, was used to label 6-aminohexanoate-dimer hydrolase, a nylon oligomer degradative enzyme of Flavobacterium sp. K172. More than 95% of the enzyme activity was lost upon incorporation of 1-1.5 molecules inhibitor/subunit of the enzyme. The tryptic peptide of the labeled enzyme was purified by HPLC (reverse-phase partition) and its amino acid sequence was identified. Radioactivity was found to be incorporated into an 8-amino-acid peptide (108His-Leu-Leu-Met-Ser-Val-Ser-Lys115). Amino acid alteration from Ser to Ala at the position 112 by site-directed mutagenesis caused loss of enzyme activity to below the detection threshold (1% of the activity of the parental enzyme). These results indicate that Ser112 is essential for the activity.     

Proteomic characterization of serine hydrolase activity and composition in normal urine

Background

Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.

Methods

Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.

Results

Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.

Conclusions

This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.     

Discovering potent and selective reversible inhibitors of enzymes in complex proteomes

To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), triacylglycerol hydrolase (TGH) and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.     

Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry

Protein expression profiles vary considerably between human cell lines and tissues, which is in part a reflection of their specialized roles within an organism. It is of considerable practical use to establish which proteins constitute the primary components of the respective proteomes. When compiled into databases, such information can facilitate the assessment of selectivity and specificity of a wide range of proteomic experiments. Here we describe the major constituents of proteomes of six human immortalized cell lines. By employing a combination of one-dimensional SDS-PAGE and nanocapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified up to 1785 non-redundant cytoplasmic and nuclear proteins from a single cell line using 50 and 30 microg of total protein from the corresponding fractions. Up to 38 proteins could be identified from a single band in one liquid chromatography-MS/MS experiment. When combined with systematic gridding of gel lanes into 48 slices, a dynamic range for protein identification of approximately 1:2000 can be envisaged for this approach. Identified proteins range from 4-553 kDa in size, cover the pI range between 3.4 and 12.8, and include 255 proteins with predicted transmembrane domains. Repeated analysis of peptides derived from the same gel band showed that the reproducibility of nanocapillary liquid chromatography-MS/MS of such complex mixtures is about 60-70% suggesting that a particular analytical experiment would need to be repeated about three times to arrive at a representative estimate of the set of highly abundant proteins in a given proteome. Given its technical simplicity, sensitivity, and wealth of generated information, we have adopted this experimental approach to characterize every cell line and tissue that is the subject of experimentation in our laboratory. The combined dataset for the six cell lines consists of 2341 non-redundant human proteins and thus constitutes one of the largest collections of human proteomic data published to date.     

Formation of the prothrombin-phosphatidyl serine and thrombin-phosphatidyl serine complex]

A possibility of formation of the thrombin-phosphatidyl serine and prothrombin-phosphatidyl serine complex is discussed. Prothrombin incubation with 131J-labelled phosphatidyl serine and its subsequent activation results in a formation of a thrombin-phosphatidyl serine--131J-complex. The radioactive label is also detected in the protein precipitate after thermodenaturation of thrombin preincubated with 131J-labelled phosphatidyl serine, which suggests that thrombin is firmly bound to phosphatidyl serine. The formation of the thrombin-phosphatidyl serine and prothrombin-phosphatidyl serine complex is supported by data of gel-filtration on Sephadex G-200 and acrylexes P-150 and P-60, as well as by differential spectrophotometry.     

Profiling protease activities by dynamic proteomics workflows

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.     

Human retinoblastoma binding protein 9, a serine hydrolase implicated in pancreatic cancers

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.     

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.     

Novel aliphatic epoxide hydrolase activities from dematiaceous fungi

Abstract Epoxide hydrolases were found to be constitutively expressed in dematiaceous fungi coincident with secondary metabolite pigment production in stationary or idiophase. Washed-cell preparations of two fungi, Ulodadium atrum CMC 3280 and Zopfiella karachiensis CMC 3284, exhibited affinity for 2,2-dialkylated oxiranes, for which contrasting enantioselectivities were observed, but not for aromatic styrene oxide or alicyclic cyclohexene oxide type substrates. Lyophilised preparations of soluble epoxide hydrolase activities proved to be effective catalysts for the mild hydrolysis of aliphatic epoxides.