Functional proteomic analysis of lipases and esterases in cultured human adipocytes

This study reports on the analysis of the lipolytic proteome of cultured human fat cells. We used specific affinity tags to detect and identify the lipolytic and esterolytic enzymes in human subcutaneous (Sc) and visceral (Visc) adipocytes. For this purpose, differentiated fat cells were incubated with a fluorescent suicide inhibitor followed by protein separation using one- or two-dimensional gel electrophoresis. After detection by fluorescence laser scanning, the labeled proteins were tryptically digested and peptides were identified by mass spectrometry. In addition, a biotinylated probe was used for specific enzyme labeling with subsequent avidin affinity isolation of the tagged proteins. Finally, we determined the quantitative differences in protein expression levels between subcutaneous and visceral adipocytes using differential activity-based gel electrophoresis (DABGE). We found that the lipase/esterase patterns of both cell types are very similar, except for some proteins that were only found in Sc cells. Two novel enzyme candidates identified in this study were overexpressed and characterized using biologically relevant glycerolipid substrates in vitro. Both of them showed pronounced hydrolytic activities on hydrophobic acylglycerols and therefore may be considered lipases. The physiological functions of the novel lipolytic proteins in vivo are currently subject to investigation.     

The lipolytic proteome of mouse adipose tissue

Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.     

Lipolytic and esterolytic activity-based profiling of murine liver

In lipid metabolism, the liver acts as a buffer for transient energy fluctuations. It temporarily stores fatty acids as triacylglycerol and secretes them as very low density lipoprotein into the circulation when the period of maximum lipid load has passed. The lipolytic enzymes responsible for mobilization of internal lipid stores in the liver have not been identified yet. We introduced active site-directed chemical probes for lipolytic activity profiling in complex mixtures, known as activity-based proteomics, and employed it for global analysis and functional annotation of lipolytic proteins in mouse adipose tissue. Here we report the combined application of two approaches using fluorescent and biotinylated probes for discovery and discrimination of lipolytic and esterolytic enzymes in mouse liver subproteomes. Proteomes labeled with the fluorescent probes were analyzed by 2-DE while proteomes labeled with the biotinylated probe were subjected to avidin-affinity isolation. Of 37 totally identified proteins, 15 were detected using both approaches while 14 and 8 were solely identified by 2-DE and avidin-affinity isolation, respectively. Moreover, 12 enzymes were classified as potential lipases and/or cholesteryl esterases by their reaction with probes specific for the respective activities directly in their proteomes.     

Synthesis activity-based zymography for detection of lipases and esterases

A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.     

Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterisation

Thermus thermophilus HB27 produces important levels of extracellular lipolytic activity when grown for 30 h at 70 degrees C in a complex medium. A method to detect esterase activity in these samples after non-reducing SDS-polyacrylamide electrophoresis was developed. The method, that implies the renaturalisation of the enzymes in the SDS-gels by washing with Triton X-100 at high temperatures, allowed detecting three esterases with different molecular weights (108, 62 and 34 kDa, respectively). The electrophoretic mobility determined under different experimental conditions suggested that the 34- and 108 kDa-esterases might correspond with two oligomeric states of a sole enzyme (monomer and trimer). Dissociation of the trimer into the monomer started when the samples were heated at temperatures higher than 60 degrees C in the presence of sodium dodecyl sulphate. Evidences were found that indicated the independent nature of the 62 kDa-esterase. A method to purify these enzymes from postincubates of T. thermophilus HB27 was developed following three steps: sodium cholate treatment, ethanol/ether precipitation and hydrophobic chromatography. In this way, an enzyme solution was obtained that contained the identified esterases/lipases. The partially purified enzymes showed an optimum of activity for the hydrolysis of p-nitrophenyl laurate at alkaline pH values and 80 degrees C, a high thermal stability and were very stable in the presence of high concentrations of isopropanol.     

Profiling serine hydrolase activities in complex proteomes

Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.     

双向凝胶电泳中三种蛋白质检测方法的比较

高通量双向电泳是蛋白质组学的核心 ,双向电泳凝胶上蛋白质点的检测方法应具有灵敏度高、线性范围宽和兼容质谱鉴定等优点 .采用差异凝胶电泳 (differencegelelectrophoresis ,DIGE)技术以Cy3(1 (5 carboxypentyl) 1′ propylindocarbocyaninehalideN hydroxysuccinimidylester)和Cy5 (1 (5 carboxypentyl) 1′ methylindodicarbocyaninehalideN hydroxysuccinimidylester)荧光分别标记正常和TNF α处理细胞的蛋白质 ,用Cy2 (3 (4 carboxymethyl)phenylmethyl) 3′ ethyloxacarbocyaninehalideN hydroxysuccinimidylester)荧光标记正常和TNF α处理细胞蛋白质的等量混合样品作为内标 ,混合 3种荧光标记的蛋白质后 ,在同一等电聚焦胶条进行聚焦 ,然后在聚丙烯酰胺凝胶上进行第二向电泳 ,用 3种波长的激光激发扫描得到凝胶图象 ,DIGE中多个样品在同一条件下电泳 ,因而匹配率高 ,且引入内标使蛋白质点的检测与定量更为准确 .DIGE技术与质谱相结合 ,实现了高通量和相对准确定量 .与硝酸银和考马斯亮蓝染色结果相比较 ,DIGE技术具有灵敏度高、线性范围宽和不影响后续质谱鉴定等优点     

嗜麦芽窄食单胞菌OUC_Est10全基因组测序及脂类水解酶多样性分析

[目的]本研究的目的是研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。[方法]使用离子交换层析、全基因组测序和异源表达三种方法研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。[结果]离子交换层析结果显示嗜麦芽窄食单胞菌OUC_Est10可以分泌多种脂类水解酶。通过全基因组测序,我们给出了该菌的全基因组序列,该基因组大小为4668743 bp,GC含量为66.25%。通过详细的基因组序列分析,我们从该基因组中找到33个可能具有脂类水解酶活性的假定基因。通过异源表达OUC_Est10中的5个假定脂类水解酶基因,来研究其催化特性的多样性,结果显示这些脂类水解酶具有不同的催化特性。[结论]我们证明了嗜麦芽窄食单胞菌OUC_Est10拥有多样的脂类水解酶,这暗示了它在不同领域中的应用潜力。     

Fluorescent organophosphonates as inhibitors of microbial lipases

Short- and long-chain 1-O-alkyl-2-acylaminodeoxyglycero- and alkoxy-alkylphosphonic acid p-nitrophenyl esters were synthesized as inhibitors for analytical and mechanistic studies on lipolytic enzymes. The respective compounds contain perylene or nitrobenzoxadiazole as reporter fluorophores covalently bound to the omega-ends of the respective 2-acylamino- and alkoxy- residues. Their inhibitory effects on the activities of three selected lipases showing different substrate preferences were determined, including the lipases from Rhizopus oryzae, Pseudomonas species, and Pseudomonas cepacia. R. oryzae lipase reacted much better with the single-chain inhibitors than the two-chain deoxyglycerolipids. In contrast, P. cepacia lipase was inactivated by perylene-containing two-chain phosphonate (XXII) to a larger extent as compared to the other inhibitors whereas Pseudomonas species lipase interacted efficiently and without any preferences with all inhibitors used in this study. In summary, the different lipases show a very characteristic reactivity pattern not only with respect to triacylglycerol substrates but also to their structurally related inhibitors. Thus, the novel phosphonates might be useful tools not only for analysis and discrimination of known lipolytic enzymes but also for discovery of yet unknown lipases/esterases in biological samples.     

Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories

Microbial metagenomes derived from soils are rich sources for the discovery of novel genes and biocatalysts. Fourteen environmental plasmid and seven fosmid libraries obtained from 10 German forest soils (A horizons) and six grassland soils (A and B horizons) were screened for genes conferring lipolytic activity. The libraries comprised approximately 29.3 Gb of cloned soil DNA. Partial activity-based screening of the constructed libraries resulted in the identification of 37 unique lipolytic clones. The amino acid sequences of the 37 corresponding lipolytic gene products shared 29-90% identity to other lipolytic enzymes, which were mainly uncharacterized or derived from uncultured microorganisms. Multiple sequence alignments and phylogenetic tree analysis revealed that 35 of the predicted proteins were new members of known families of lipolytic enzymes. The remaining two gene products represent two putatively new families. In addition, sequence analysis indicated that two genes encode true lipases, whereas the other genes encode esterases. The determination of substrate specificity and chain-length selectivity using different triacylglycerides and p-nitrophenyl esters of fatty acids as substrates supported the classification of the esterases.     

Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes

We report on the determination of active enzyme components in pure and crude lipases, using fluorescent inhibitors for covalent modification and visualization of the enzymatically active proteins. Lipase-specific compounds are triacylglycerol analogs, namely 1,2(2, 3)-di-O-alkylglyceroalkylphosphonic acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcohols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible toward nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protein complexes are formed which can be analyzed on the basis of their fluorescent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the analysis of a commercial lipase preparation from Rhizomucor miehei. After incubation of the enzyme with the fluorescent lipid, a single fluorescence band was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtained between fluorescence intensity and the amount of enzyme. Using a combination of different inhibitors, we were able to discriminate between lipases and esterases.     

Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome

Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases, EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids in length, respectively, and showed the highest similarity to class C β-lactamases. However, they shared a relatively low level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1 and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)₆-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0–35°C, with optimal temperatures of 20°C and 30°C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The β-lactamase activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus β-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential biotechnological applications as interesting biocatalysts.     

Sub-proteome differential display: single gel comparison by 2D electrophoresis and mass spectrometry

Two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) have been used in comparative proteomics but inherent problems of the 2D electrophoresis technique lead to difficulties when comparing two samples. We describe a method (sub-proteome differential display) for comparing the proteins from two sources simultaneously. Proteins from one source are mixed with radiolabelled proteins from a second source in a ratio of 100:1. These combined proteomes are fractionated simultaneously using column chromatographic methods, followed by analysis of the pre-fractionated proteomes (designated sub-proteomes) using 2D gel electrophoresis. Silver staining and (35)S autoradiography of a single gel allows precise discrimination between members of each sub-proteome, using commonly available computer software. This is followed by MS identification of individual proteins. We have demonstrated the utility of the technology by identifying the product of a transfected gene and several proteins expressed differentially between two renal carcinoma proteomes. The procedure has the capacity to enrich proteins prior to 2D electrophoresis and provides a simple, inexpensive approach to compare proteomes. The single gel approach eliminates differences that might arise if separate proteome fractionations or 2D gels are employed.     

Esterase EstA6 from Pseudomonas sp. CR-611 is a novel member in the utmost conserved cluster of family VI bacterial lipolytic enzymes

Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.     

GDSL family of serine esterases/lipases

GDSL esterases and lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of important ester compounds of pharmaceutical, food, biochemical, and biological interests. This new subclass of lipolytic enzymes possesses a distinct GDSL sequence motif different from the GxSxG motif found in many lipases. Unlike the common lipases, GDSL enzymes do not have the so called nucleophile elbow. Studies show that GDSL hydrolases have a flexible active site that appears to change conformation with the presence and binding of the different substrates, much like the induced fit mechanism proposed by Koshland. Some of the GDSL enzymes have thioesterase, protease, arylesterase, and lysophospholipase activity, yet they appear to be the same protein with similar molecular weight (22–60 kDa for most esterases), although some have multiple glycosylation sites with higher apparent molecular weight. GDSL enzymes have five consensus sequence (I–V) and four invariant important catalytic residues Ser, Gly, Asn, and His in blocks I, II, III, and V, respectively. The oxyanion structure led to a new designation of these enzymes as SGNH-hydrolase superfamily or subfamily. Phylogenetic analysis revealed that block IIA which belonged to the SGNH-hydrolases was found only in clade I. Therefore, this family of hydrolases represents a new example of convergent evolution of lipolytic enzymes. These enzymes have little sequence homology to true lipases. Another important differentiating feature of GDSL subfamily of lipolytic enzymes is that the serine-containing motif is closer to the N-terminus unlike other lipases where the GxSxG motif is near the center. Since the first classification of these subclass or subfamily of lipases as GDSL(S) hydrolase, progress has been made in determining the consensus sequence, crystal structure, active site and oxyanion residues, secondary structure, mechanism of catalysis, and understanding the conformational changes. Nevertheless, much still needs to be done to gain better understanding of in vivo biological function, 3-D structure, how this group of enzymes evolved to utilize many different substrates, and the mechanism of reactions. Protein engineering is needed to improve the substrate specificity, enantioselectivity, specific activity, thermostability, and heterologous expression in other hosts (especially food grade microorganisms) leading to eventual large scale production and applications. We hope that this review will rekindle interest among researchers and the industry to study and find uses for these unique enzymes.     

激素型肾阳虚动物肝线粒体蛋白质组与能量代谢相关性

应用凝胶内差异显示电泳技术研究肾阳虚大鼠肝线粒体蛋白质组,并从肝线粒体蛋白质组角度阐述肾阳虚与能量代谢的关系.8个分别来自于肾阳虚大鼠和正常大鼠的肝线粒体蛋白质样品(各4个)分别用荧光染料Cy3、Cy5标记,以及8个样品等量混合物用Cy2标记作为内标,每一Cy3、Cy5标记样品与Cy2标记的内标等量混合后在同一胶中进行电泳分离,经不同光激发后扫描得到不同样品的蛋白质组图谱.经DeCyder软件结合内标分析,以肾阳虚组动物与正常组动物肝线粒体蛋白质相差1.2倍以上的蛋白作为差异蛋白,实验共获得16个差异蛋白质,经质谱测定和与蛋白质文库比对,鉴定11个蛋白质.其中,肾阳虚动物热休克蛋白60和70、肌氨酸脱氢酶、氨甲酰磷酸合成酶、亚硫酸盐氧化酶、ATP合酶、醛脱氢酶和NADH脱氢酶表达量增加,而丙酮酸脱氢酶、α酮戊二酸脱氢酶、脂酰辅酶A脱氢酶和鸟氨酸氨基转移酶表达量降低.实验表明,肾阳虚动物能量代谢相关酶的变化与肾阳虚的临床虚寒症状有关.     

Activity based subcellular resolution imaging of lipases

Lipases play a key role in whole body energy homeostasis. Dysregulation of lipolytic activities affects lipid absorption, mobilization, and transport, and is causative for lipid-related diseases. Regulation of enzymes involved in lipid metabolism is governed by a complex network of protein-protein and protein-small molecule interactions. Thus these enzymes have to be studied under the physiologically most relevant conditions, that is, in vivo. Our latest generation of activity based probes designed for capturing of lipases employs bioorthogonal chemical linker groups, which are membrane permeable and thus allow studying protein activity in living cells. Another advantage is the virtually unlimited choice of reporter tags. Here we report on a novel method combining in vivo activity based labeling of lipases with in situ detection of lipolytic activities by on slide click chemistry and imaging by fluorescence microscopy. We demonstrate that cytosolic as well as organelle resident lipases are specifically labeled in intact living cells. This method will shed light on the (sub)cellular localization of lipolytic proteomes of cells and tissues in health and disease directly at enzymatic activity level without the need of prior knowledge of the identities of the responsible enzymes or dependence on the availability of specific antibodies.     

Polymer membrane ion-selective electrodes as a convenient tool for lipases and esterases assays

We have proposed a novel assay for lipases and esterases activity determination based on potentiometry with ion-selective electrodes (ISEs). Enzyme preparations, obtained from the living cells, are complex mixtures of various proteins, short peptides, lipids, carbohydrates, and other compounds. The most commonly used quantitative methods in enzyme studies are based on spectrophotometric or spectroflourimetric protocols which has significant limitations. They are not valid for samples that are turbid or strongly colored. To overcome those drawbacks we have proposed an assay based on potentiometry with ISEs for lipases and esterases activity determination. This electrochemical methodology represents an attractive tool for enzyme analysis, because of its low detection limit, independence from sample volume and from sample turbidity. The usefulness of this assay has been proven by the determination of the activity of various raw enzymes “acetone powders” isolated from animal tissues. Moreover, activities of fractions obtained during purification of one of those raw biocatalysts were also determined that way. The reliability of determination enzyme activity with ISE assay was proven by comparison with a classical spectrophotometric method.     

Enzymatic profile of Toxoplasma gondii

Zymogram patterns of nine strains of Toxoplasma gondii were studied using the API enzyme research kit. This system uses chromogenic substrates to detect the presence or absence of 84 enzymes. Enzyme classes assayed for included amino-peptidases, glycosidases, esterases, lipases, phosphoamidase and phosphatases.
All strains were positive for 24 enzymes: 15 arylamidases, seven esterases, alkaline phosphatase and acid phosphatase. In contrast, 27 enzyme tests were negative in all strains. Thirty-three enzymatic reactions were different in one or more strains.     

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.