Activity-based Protein Profiling of Serine Hydrolase Superfamily Enzymes.

Activity-based protein profiling (ABPP) is a chemoproteomics platform to assess the functional state of enzymes in complex biological systems. Over the two decades, ABPP has emerged from a gel-based to gel-free platform, for in-depth proteome analysis with enhanced resolution, sensitivity for target detection, and discovery of small molecule inhibitors. The gel-free format of ABPP coupled with advanced mass spectrometry is highly sensitive and provides more comprehensive knowledge for the targeted enzyme family than the gel-based method. ABPP strategy is applied across microbe, plant, and animal models. It can be performed both in vitro and in vivo studies, and there is no limitation on sample origin. Here, we report an ultrasensitive, gel-free format of ABPP called active site peptide profiling. This protocol describes the identification of authentic functional proteins, by tagging their active sites in a native biological system. It is high throughput in nature and helps enrich even low abundance functional proteins. Since protein identification is virtually based on a single peptide, the identified peptide should be a unique peptide to identify its parent protein. It can be performed in a facile manner and offers to consolidate identification of protein targets as well as the site of probe modification. We have validated this approach using a fluorophosphonate (FP) serine hydrolase probe in the native proteome of the cereal crop Oryza sativa. Graphic abstract: Serine hydrolase active site peptide profiling.

Proteoform of Arabidopsis seed storage protein identified by functional proteomics approach exhibits acyl hydrolase activity during germination.

Lipases play a crucial role in the life cycle of seed plants and the oil content of the seed is highly regulated by the lipase activity. Hence, understanding the role of lipases during germination and post-germination will provide insights into lipid mobilization. However, to date, no lipase gene has been identified in seeds except, Sugar-dependent-1 in Arabidopsis. Hence, in the present study, we employed a functional proteomic approach for the identification of seed-specific lipase. Activity-Based Proteome Profiling (ABPP) of Arabidopsis mature and germinating seeds revealed the expression of a functional serine hydrolase exclusively during germination. The mass-spectrometry analysis reveals the identity and amino acid sequence of the protein correspond to AT4G28520 gene, a canonical 12S Seed Storage Protein (SSP). Interestingly, the identified SSP was a proteoform of AT4G28520 (SL-AT4G28520) and exhibited >90% identity with the canonical AT4G28520 (FL-AT4G28520). Heterologous expression and enzyme assays indicated that SL-AT4G28520 protein indeed possesses monoacylglycerol lipase activity, while the FL-AT4G28520 protein didn't exhibit any detectable activity. Functional proteomics and lipidomics analysis demonstrated a catalytic function of this SSP. Collectively, this is the first report, which suggests that SL-AT4G28520 encodes a lipase, and the activity is depending on the physiological condition.

Lipidome, nutraceuticals and nutritional profiling of Pyrus pashia Buch.-ham ex D. Don (Kainth) seeds oil and its antioxidant potential.

Kainth fruit, as traditional medicine, has been used in the Himalayan region for its health-promoting properties. However, the phytochemicals and lipidomes of Kainth Seed Oil (KSO) are still scarce. Here, we investigated the physicochemical characterization of KSO and its nutraceuticals, antioxidant potentials. Kainth seeds contain 19-20% oil rich in polyunsaturated fatty acids (PUFA, 82.22%), particularly linoleic acid (C18:2). Lipidome analysis of KSO using high-resolution mass spectrometry showed that trilinoleate (C54:6) was the dominant triacylglycerol (TAG) species. Further, the characteristics of PUFA-rich oil were validated by Fourier transforms infrared spectroscopy (FTIR) and Differential Scanning calorimetry (DSC). The nutraceuticals profiling of KSO depicted the presence of tocopherols (86.72 mg) and phytosterols (32.25 mg) in 100 g oil with significant antioxidant activity. The oil cake contained 19.09% protein and minerals and can be a source for dietary protein. Collectively these results suggest that KSO will be a suitable source for PUFA and nutraceuticals potential.

Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases.

Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. The endogenous lipases and hydrolases are responsible for the rapid deterioration of rice bran. Hence, we attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. The active site-directed fluorophosphonate probe (rhodamine and biotin-conjugated) was used for the detection and identification of active SHs. ABPP revealed 55 uncharacterized active-SHs and are representing five different known enzyme families. Based on motif and domain analyses, one of the uncharacterized and miss annotated SHs (Os12Ssp, storage protein) was selected for biochemical characterization by overexpressing in yeast. The purified recombinant protein authenticated the serine protease activity in time and protein-dependent studies. Os12Ssp exhibited the maximum activity at a pH between 7.0 and 8.0. The protease activity was inhibited by the covalent serine protease inhibitor, which suggests that the ABPP approach is indeed reliable than the sequence-based annotations. Collectively, the comprehensive knowledge generated from this study would be useful in expanding the current understanding of rice bran SHs and paves the way for better utilization/stabilization of rice bran.

Functional Omics Identifies Serine Hydrolases That Mobilize Storage Lipids during Rice Seed Germination.

Elucidating proteolipidome dynamics is crucial for understanding the roles of these molecules in plant physiology and disease. Sequence-based functional annotation of the protein is inadequate, since protein activities depend on posttranslational modification. In this study, we applied a gel-free activity-based protein profiling approach to unravel the active lipases, including other Serine hydrolases (SHs), expressed during seed germination in rice (Oryza sativa). We successfully mapped the active sites of 43 active SHs encompassing lipases/esterases, GDSL lipases, proteases, Ser carboxypeptidases, ABHD protein, pectin acetylesterase, and other SHs. The mRNA expression levels of those genes encoding the identified SHs were monitored using microarray analysis. The lipidome analysis revealed distinct patterns of molecular species distribution in individual lipid classes and displayed the metabolic connections between lipid mobilization and rice seedling growth. Changes in the mobilization of storage lipids and their molecular species remodeling were correlated with the expression of the identified lipases and their lipase activity in a time-dependent manner. The physiological significance of the identified SHs was explored during biotic stress with Fusarium verticillioides infection. The fungal infection significantly reduced lipase activity and lipid mobilization, thus impairing the rice seedling. Collectively, our data demonstrate application of the functional proteome strategy along with the shotgun lipidome approach for the identification of active SHs, and thus for deciphering the role of lipid homeostasis during rice seed germination.

A solvent-free delipidation method for functional validation of lipases.

Extracting protein in its active form is critical for its functional characterization, and lipid removal is an essential step in the protein extraction process for further downstream applications. In the present study, we revisited the delipidation protocol and developed a rapid, solvent-free delipidation method using activated silica. The delipidated samples showed improved optical clarity and a significant reduction of endogenous lipids. The functional integrity of the lipases present in the delipidated sample was validated by in vitro enzyme assay using physiological substrate which includes neutral lipid as well as phospholipid. The accessibility of active site of the extracted enzymes was demonstrated by activity-based protein profiling (ABPP), a functional chemoproteomic approach. Detection of serine hydrolases using ABPP probe labeling was enhanced upon delipidation. Further, the total polyphenol content was significantly reduced, which helps to enhance the protein enrichment and small-molecule inhibitor screening by ABPP. Collectively, these results suggest that the present solvent-free delipidation approach is efficient and highly compatible with the functional characterization of the enzymes, particularly lipid hydrolases.

OsPLB gene expressed during seed germination encodes a phospholipase in rice.

Hydrolysis of phospholipid monolayer by phospholipases is an important event in the mobilization of stored lipids for seed germination. However, the identification and functional characterization of cereal phospholipases, especially during rice germination, are limited. In the present study, we have identified and characterized a phospholipase OsPLB gene expressed during germination. The full-length coding region of OsPLB was cloned into pRSETA as well as pYES2/NTC vector. The recombinant protein was successfully expressed in both E. coli and Saccharomyces cerevisiae. The recombinant protein was purified to homogeneity by affinity chromatography, and it was further confirmed by MS/MS analysis. In vitro lipase assay and lipidome analysis using high-resolution mass spectrometry showed phosphatidylcholine (PC) specific phospholipase B activity. The results revealed that protein encoded by OsPLB gene prefers to hydrolyze PCs with C28, C32, and C34 containing unsaturated fatty acids. Collectively, the present study describes the identification and characterization of a phospholipase B, which hydrolyze PC, a major component of phospholipid monolayer covering storage lipid, as an initial event during rice seed germination.

Cyanidin and cyanidin-3-glucoside derived from Vigna unguiculata act as noncompetitive inhibitors of pancreatic lipase.

The consumption of legumes positively correlated with the reduction of body weight. In the present study, we identified and evaluated pancreatic lipase inhibitors from Vigna unguiculata and unraveled their mode of inhibition. The highly sensitive fluorometric method was adopted to access the pancreatic lipase activity and the ethanolic extract of Vigna unguiculata showed the maximum inhibition (IC50 of 15.2 µg/ml). Cyanidin and cyanidin-3-glucoside are the major anthocyanins observed in Vigna unguiculata. The IC50 value of cyanidin was 28.29 µM which was 6.5-fold higher than the cyanidin-3-glucoside (188.28 µM). We determined an apparent Ki of 27.28 µM for cyanidin and cyanidin-3-glucoside (88.97 µM) with noncompetitive inhibition. Collectively, these results suggest that the glycosylation of the anthocyanidins significantly reduces lipase inhibition. The noncompetitive inhibition of pancreatic lipase by Vigna unguiculata anthocyanins may exert significant pharmacological activities toward obesity complications by calorie restriction. PRACTICAL APPLICATIONS: The results of this study emphasize the importance of legumes in our diet to combat obesity-related complications. Consumption of legumes minimizes fat absorption by inhibiting the action of the fat-digesting enzyme.

The Arabidopsis ABHD11 Mutant Accumulates Polar Lipids in Leaves as a Consequence of Absent Acylhydrolase Activity.

Alpha/beta hydrolase domain (ABHD)-containing proteins are structurally related with diverse catalytic activities. In various species, some ABHD proteins have been characterized and shown to play roles in lipid homeostasis. However, little is known about ABHD proteins in plants. Here, we characterized AT4G10030 (AtABHD11), an Arabidopsis (Arabidopsis thaliana) homolog of a human ABHD11 gene. In silico analyses of AtABHD11 revealed homology with other plant species with a conserved GXSXG lipid motif. Interestingly, Arabidopsis abhd11 mutant plants exhibited an enhanced growth rate compared with wild-type plants. Quantitative analyses of the total lipids showed that the mutant abhd11 has a high amount of phospholipid and galactolipid in Arabidopsis leaves. The overexpression of AtABHD11 in Escherichia coli led to a reduction in phospholipid levels. The bacterially expressed recombinant AtABHD11 hydrolyzed lyso(phospho)lipid and monoacylglycerol. Furthermore, using whole-genome microarray and real-time PCR analyses of abhd11 and wild-type plants, we noted the up-regulation of MGD1, -2, and -3 and DGD1. Together, these findings suggested that AtABHD11 is a lyso(phospho)lipase. The disruption of AtABHD11 caused the accumulation of the polar lipids in leaves, which in turn promoted a higher growth rate compared with wild-type plants.

In vitro exposure of tobacco specific nitrosamines decreases the rat lung phospholipids by enhanced phospholipase A2 activity.

Tobacco-specific nitrosamines (TSNA) have implications in the pathogenesis of various lung diseases and conditions are prevalent even in non-smokers. N-nitrosonornicotine (NNN) and 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are potent pulmonary carcinogens present in tobacco product and are mainly responsible for lung cancer. TSNA reacts with pulmonary surfactants, and alters the surfactant phospholipid. The present study was undertaken to investigate the in vitro exposure of rat lung tissue slices to NNK or NNN and to monitor the phospholipid alteration by [(32)P]orthophosphate labeling. Phospholipid content decreased significantly in the presence of either NNK or NNN with concentration and time dependent manner. Phosphatidylcholine (PC) is the main phospholipid of lung and significant reduction was observed in PC ∼61%, followed by phosphatidylglycerol (PG) with 100µM of NNK, whereas NNN treated tissues showed a reduction in phosphatidylserine (PS) ∼60% and PC at 250µM concentration. The phospholipase A2 assays and expression studies reveal that both compounds enhanced phospholipid hydrolysis, thereby reducing the phospholipid content. Collectively, our data demonstrated that both NNK and NNN significantly influenced the surfactant phospholipid level by enhanced phospholipase A2 activity.

Effect of lipopolysaccharide on alteration of phospholipids and their fatty acid composition in spleen and thymus by in vitro metabolic labeling.

Lipopolysaccharide (LPS) is an endotoxin, a potent stimulator of immune response and induction of LPS leads to acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). ARDS is a life-threatening disease worldwide with a high mortality rate. The immunological effect of LPS with spleen and thymus is well documented; however the impact on membrane phospholipid during endotoxemia has not yet been studied. Hence we aimed to investigate the influence of LPS on spleen and thymus phospholipid and fatty acid composition by [(32) P]orthophosphate labeling in rats. The in vitro labeling was carried out with phosphate-free medium (saline). Time course, LPS concentration-dependent, pre- and post-labeling with LPS and fatty acid analysis of phospholipid were performed. Labeling studies showed that 50 µg LPS specifically altered the major phospholipids, phosphatidylcholine and phosphatidylglycerol in spleen and phosphatidylcholine in thymus. Fatty acid analysis showed a marked alteration of unsaturated fatty acids/saturated fatty acids in spleen and thymus leading to immune impairment via the fatty acid remodeling pathway. Our present in vitro lipid metabolic labeling study could open up new vistas for exploring LPS-induced immune impairment in spleen and thymus, as well as the underlying mechanism.

Antihyperlipidemic activity of Cassia auriculata flowers in triton WR 1339 induced hyperlipidemic rats.

The flower extract of Cassia auriculata, herb has been used traditionally in India for medicinal purposes. The plant has been reported to treat hyperglycemia and associated hyperlipidemia. Hyperlipidemia and oxidative stress are known to accelerate coronary artery disease and progression of atherosclerotic lesions. The present work was undertaken to investigate the possible antihyperlipidemic and antioxidative effect of C. auriculata flower on hyperlipidemic rats. Hyperlipidemia was induced in rats by a single intravenous (iv) injection of Triton WR 1339 (300 mg/kg b.w.) and it showed sustained elevated levels of serum cholesterol and triglyceride. Ethanolic extract of C. auriculata flowers (Et-CAF) (150, 300, 450 mg/kg b.w./day) was administered to normal and hyperlipidemic rats for 14 days. Serum and liver tissue were analysed at three different time intervals for lipid profile, lipid peroxidation products, antioxidants enzymes and the activity were compared to the cholesterol-lowering drug, lovastatin (10 mg/kg/b.w.). Parameters were altered during hyperlipidemia and reverted back to near normal values after Et-CAF treatment or standard drug lovastatin. Lipid peroxidation decreased whereas the activities of superoxide dismutase, glutathione peroxidase and catalase increased in Et-CAF treated rats. Pronounced changes were observed at 450 mg/kg b.w. of Et-CAF for 2 weeks and it was comparable to the standard drug lovastatin. The current study provides a strong evidence that Et-CAF has a beneficial effect in treating hyperlipidemia and ROS without any side effects at the dosage and duration studied.

A bifunctional enzyme that has both monoacylglycerol acyltransferase and acyl hydrolase activities.

Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX(4)D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions.

Enhanced phospholipase B activity and alteration of phospholipids and neutral lipids in Saccharomyces cerevisiae exposed to N-nitrosonornicotine.

A tobacco-specific nitrosamine (TSNA), N-nitrosonornicotine (NNN), is a potent carcinogen present in cigarette smoke, and chronic exposure to it can lead to pulmonary cancer. NNN causes changes in phospholipid metabolism and the mechanism is yet to be elucidated. Exposure of Saccharomyces cerevisiae to 50 µM NNN leads to a substantial decrease in phosphatidylserine (PS) by 63%, phosphatidylcholine (PC) by 42% and phosphatidylethanolamine (PE) by 36% with a concomitant increase in lysophospholipids (LPL) by 25%. The alteration in phospholipid content was dependent on increasing NNN concentration. Reduced phospholipids were accompanied with increased neutral lipid content. Here we report for the first time that NNN exposure, significantly increases phospholipase B (PLB) activity and the preferred substrate is PC, a major phospholipid responsible for a series of metabolic functions. Furthermore, NNN also promotes the alteration of fatty acid (FA) composition; it increases the long chain fatty acid (C18 series) in phospholipids specifically phosphatidylethanolamine (PE) and PS; while on the contrary it increases short chain fatty acids in cardiolipin (CL). NNN mediated degradation of phospholipids is associated with enhanced PLB activity and alteration of phospholipid composition is accompanied with acyl chain remodelling. Understanding the altered phospholipid metabolism produced by NNN exposure is a worthwhile pursuit because it will help to understand the toxicity of tobacco smoke.

Induction of acute respiratory distress syndrome in rats by lipopolysaccharide and its effect on oxidative stress and antioxidant status in lung.

Acute lung injury (ALI) or its severe form, acute respiratory distress syndrome (ARDS) is an important cause of mortality in the human population. Despite significant advances made, the mortality associated with ALI remains unchanged. The objective of the present study was to evaluate the role of oxidative stress, alveolar antioxidant status and multiple organ injury in ARDS induced by lipopolysaccharide (LPS) in rats. Rats were divided into 4 groups, group I control rats were given saline intraperitoneally, whereas groups II, III and IV (LPS-treated) rats received an intraperitoneal injection of LPS (10 mg/kg body weight) and sacrificed after various time intervals. In LPS-treated rats, we observed increased levels of oxidative products, decreased levels of antioxidants in lung tissues and increased levels of serum marker enzymes, suggesting multiple organ injury. Bronchoalveolar lavage fluid (BALF) neutrophil content and protein concentration in LPS-treated rats were significantly elevated in a time-dependent manner. Histological studies revealed neutrophil influx and diffused alveolar damage in LPS-administered rats. These results clearly suggested that increased oxidant levels led to oxidative stress, antioxidant deficiency attenuating lung inflammation and tissue damage. LPS administration resulted in multiple organ failure, leading to increased mortality.