Endothelial lipase: a new member of the triglyceride lipase gene family

The triglyceride lipase gene family plays a central role in intestinal lipid absorption, energy homeostasis, lipoprotein metabolism, and atherosclerosis. A new member of this gene family, termed endothelial lipase, was recently reported. The presence of key functional motifs, the endothelial synthesis, the enzymatic profile, and the in-vivo metabolic effects of endothelial lipase suggest that, like other members of this gene family, endothelial lipase may play a role in energy delivery to tissues and in modulating lipoprotein metabolism, and could impact on atherogenesis.     

Optimization of nucleic acid sequences

Base sequence influences the structure, mechanics, dynamics, and interactions of nucleic acids. However, studying all possible sequences for a given fragment leads to a number of base combinations that increases exponentially with length. We present here a novel methodology based on a multi-copy approach enabling us to determine which base sequence favors a given structural change or interaction via a single energy minimization. This methodology, termed ADAPT, has been implemented starting from the JUMNA molecular mechanics program by adding special nucleotides, "lexides," containing all four bases, whose contribution to the energy of the system is weighted by continuously variable coefficients. We illustrate the application of this approach in the case of double-stranded DNA by determining the optimal sequences satisfying structural (B-Z transition), mechanical (intrinsic curvature), and interaction (ligand-binding) properties.     

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

A comparison of the cDNA of the sequences of signal receptor proteins]

The method of cDNA sequences comparison is suggested. The method consists of constructing the dot matrixes for each codon position separately. The fastest disappearance of the information about relationship is observed when the third nucleotides in codon are compared, and the comparison of the second nucleotides in codon turned out to be effective so as the comparison under acids.     

Graphical coding of nucleic acid sequences

When, in a nucleic acid sequence, the four letters C, G, A, T (or U) are replaced by suitable graphical symbols, some patterns become immediately apparent. Two sets of symbols, constructed for the analysis of either purine/pyrimidine alternations, or of regions of complementarity within a sequence are shown. In addition, another mode of coding is presented, in which the four letters are represented by vectors. The sequence is thus transformed into a planar trajectory. We show, in the case of the gene for human beta hemoglobin, that such a coding enables an easy discrimination between introns and exons.     

Rational recognition of nucleic acid sequences

Recent progress in synthetic and computational chemistry has made it possible to develop certain novel drug candidates. Drug candidates for genetic diseases, such as cancer, may also be designed on the basis of structural information obtained using X-ray analysis and NMR, as well as evidence from biological techniques applied to natural products - DNA (or RNA) complexes and conjugates. The resulting designed drug candidates exhibit promising performance based on the recognition on nucleic acid sequences.     

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a Proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5 kDa) and 347 aa (38.6 kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

JaDis: computing distances between nucleic acid sequences.

SUMMARY: JaDis is a Java application for computing evolutionary distances between nucleic acid sequences and G+C base frequencies. It allows specific comparison of coding sequences, of non-coding sequences or of a non-coding sequence with coding sequences. AVAILABILITY: http://pbil.univ-lyon1.fr/software/jadis.html     

Homology of Drosophila yolk proteins and the triacylglycerol lipase family

Drosophila yolk proteins consist of a set of related proteins of 50,000 Mr. They are derived from slightly larger precursors by cleavage of a signal peptide. In this respect, they differ from the yolk proteins of other insects which are proteolytic fragments of precursors of 200,000 Mr or larger, termed vitellogenins and probably homologous to the vitellogenins of other egg-laying species. We report here a comparative amino acid analysis demonstrating that the Drosophila yolk proteins are non-homologous to the vitellogenin group of yolk proteins, but surprisingly are related to the triacylglycerol lipase family.     

Cloning of a unique lipase from endothelial cells extends the lipase gene family.

A new lipoprotein lipase-like gene has been cloned from endothelial cells through a subtraction methodology aimed at characterizing genes that are expressed with in vitro differentiation of this cell type. The conceptual endothelial cell-derived lipase protein contains 500 amino acids, including an 18-amino acid hydrophobic signal sequence, and is 44% identical to lipoprotein lipase and 41% identical to hepatic lipase. Comparison of primary sequence to that of lipoprotein and hepatic lipase reveals conservation of the serine, aspartic acid, and histidine catalytic residues as well as the 10 cysteine residues involved in disulfide bond formation. Expression was identified in cultured human umbilical vein endothelial cells, human coronary artery endothelial cells, and murine endothelial-like yolk sac cells by Northern blot. In addition, Northern blot and in situ hybridization analysis revealed expression of the endothelial-derived lipase in placenta, liver, lung, ovary, thyroid gland, and testis. A c-Myc-tagged protein secreted from transfected COS7 cells had phospholipase A1 activity but no triglyceride lipase activity. Its tissue-restricted pattern of expression and its ability to be expressed by endothelial cells, suggests that endothelial cell-derived lipase may have unique functions in lipoprotein metabolism and in vascular disease.     

Metallo-beta-lactamase fold within nucleic acids processing enzymes: the beta-CASP family

A separate family of enzymes within the metallo-β-lactamase fold comprises several important proteins acting on nucleic acid substrates, involved in DNA repair (Artemis, SNM1 and PSO2) and RNA processing [cleavage and polyadenylation specificity factor (CPSF) subunit]. Proteins of this family, named β-CASP after the names of its representative members, possess specific features relative to those of other metallo-β-lactamases, that are concentrated in the C-terminal part of the domain. In this study, using sensitive methods of sequence analysis, we identified highly conserved amino acids specific to the β-CASP family, some of which were unidentified to date, that are predicted to play critical roles in the enzymatic function. The identification and characterisation of all the extant, detectable β-CASP members within sequence databases and genome data also allowed us to unravel particular sequence features which are likely to be involved in substrate specificity, as well as to describe new but as yet uncharacterised members which may play critical roles in DNA and RNA metabolism.     

Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.

A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.     

In vitro selection of functional nucleic acid sequences

The power of in vitro selection methods for the isolation of nucleic acids that display a desired property derives from the enormous number of sequence variants that can be surveyed with relative ease using controlled in vitro biochemistry. This methodology has found a variety of applications, ranging from the study of nucleic acid-protein interactions and natural ribozymes to the isolation of nucleic acids with potential as diagnostic or therapeutic reagents or with new catalytic activities. The number of reported applications is growing exponentially, and each application presents new variables and challenges. The goal of this article is to guide prospective users through the myriad decisions that must be made in the design and execution of a successful in vitro selection experiment.     

Enzyme promiscuity in the hormone-sensitive lipase family of proteins

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

Recognition of ill-defined signals in nucleic acid sequences

A set of programs has been developed for the definition andhandling of nucleic acid sequence consensus information. Thesequences of known genetic control signals are combined in amatrix. The origins and positions of the signals are recorded.Old matrices can be updated dynamically: new signals are includedand obsolete ones deleted. Matrices of several different typesare computed optionally. Several of these matrices can be combinedto find possible new signals. The use of matrices allows theexact quantification of signal qualities. The described programsare part of a program library named GENEXPERT. Application examplesgiven are the search for tRNA genes and the search for promotersin the bacteriophage genome. Received on July 22, 1987; accepted on October 5, 1987     

Calculation of folding energies of single-stranded nucleic acid sequences: conceptual issues

The stability of a folded single-stranded nucleic acid depends on the composition and order of its constituent bases and may be assessed by taking into account the pairing energies of its constituent dinucleotides. To assess the possible biological significance of a computed structure, Maizel and coworkers in the 1980s compared the energy of folding of a natural single-stranded RNA sequence with the energies of several versions of the same sequence produced by shuffling base order. However, in the 2000s many took as self-evident the view that shuffling at the mononucleotide level (single bases) was conceptual wrong and should be replaced by shuffling at the level of dinucleotides (retaining pairs of adjacent bases). Folding energies then became indistinguishable from those of corresponding shuffled sequences and doubt was cast on the importance of secondary structures. Nevertheless, some continued productively to employ the single base shuffling approach, the justification for which is the topic of this paper. Because dinucleotide pairing energies are needed to calculate structure, it does not follow that shuffling should not disrupt dinucleotides. Base shuffling allows determination of the relative contributions of base composition and base order to total folding energy. The potential for secondary structure arises from pressures acting at both DNA and RNA levels, and is abundant throughout genomes-with a probable primary role in recombination. Within a gene the potential can often be accommodated, and base order and composition work together (values have the same negative sign) in contributing to total folding energy. But sometimes protein-coding pressure on base order conflicts with the pressure for secondary structure and the values have opposite signs. Total folding energy can be deemed of potential biological significance when the average of several readings is significantly less than zero.