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T Osterlund

Publications and source records attributed to T Osterlund.

11 recordsLinked to original sources
PubMed2001

Structure-function relationships of hormone-sensitive lipase.

Research into the structure-function relationships of lipases and esterases has increased significantly during the past decade. Of particular importance has been the deduction of several crystal structures, providing a new basis for understanding these enzymes. The generated insights have, together with cloning and expression, aided studies on structure-function relationships of hormone-sensitive lipase (HSL). Novel phosphorylation sites have been identified in HSL, which are probably important for activation of HSL and lipolysis. Functional and structural analyses have revealed features in HSL common to lipases and esterases. In particular, the catalytic core with a catalytic triad has been unveiled. Furthermore, the investigations have given clear suggestions with regard to the identity of functional and structural domains of HSL. In the present paper, these studies on HSL structure-function relationships and short-term regulation are reviewed, and the results presented in relation to other discoveries in regulated lipolysis.

Adipocytes↗
PubMed2000

Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.

Hormone-sensitive lipase, the rate-limiting enzyme of intracellular TG hydrolysis, is a major determinant of fatty acid mobilization in adipose tissue as well as other tissues. It plays a pivotal role in lipid metabolism, overall energy homeostasis, and, presumably, cellular events involving fatty acid signaling. Detailed knowledge about its structure and regulation may provide information regarding the pathogenesis of such human diseases as obesity and diabetes and may generate concepts for new treatments of these diseases. The current review summarizes the recent advances with regard to hormone-sensitive lipase structure and molecular mechanisms involved in regulating its activity and lipolysis in general. A summary of the current knowledge regarding regulation of expression, potential involvement in lipid disorders, and role in tissues other than adipose tissue is also provided.

Adipose Tissue↗
PubMed1999

Domain identification of hormone-sensitive lipase by circular dichroism and fluorescence spectroscopy, limited proteolysis, and mass spectrometry.

Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Osterlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411-420; Contreras, J. A., Karlsson, M., Osterlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426-31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1-4 and 5-9, respectively, of which the latter contains an exposed regulatory module outside the catalytic alpha/beta-hydrolase fold core.

Animals↗
PubMed1999

Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase.

Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct sequence, inferred from the experimentally phased electron density map, revealed that BFAE is a homolog of the mammalian hormone sensitive lipase (HSL). It is a canonical alpha/beta hydrolase with two insertions forming the substrate binding pocket. The enzyme contains a lipase-like catalytic triad, Ser 202, Asp 308 and His 338, consistent with mutational studies that implicate the homologous Ser 424, Asp 693 and His 723 in the catalytic triad in human HSL.

Amino Acid Sequence↗
PubMed1998

Human hormone-sensitive lipase: expression and large-scale purification from a baculovirus/insect cell system.

Hormone-sensitive lipase (HSL) is a key enzyme in lipid metabolism and overall energy homeostasis in mammals. It catalyzes the rate-limiting step in the hydrolysis of triglyceride stores in the adipocytes, delivering free fatty acids for their use as energy substrates. HSL activity is under acute hormonal and neural control, mediated through reversible phosphorylation of the enzyme. Emerging data from clinical studies indicate that HSL deficiency or malfunction is associated with several pathological situations in humans. In order to perform a biochemical characterization of human HSL, and to elucidate its molecular properties, purification of homogeneous protein in large amounts is required. Here, we describe the expression and purification of a catalytically active recombinant human HSL. The process allows the purification of milligram amounts of homogeneous protein, and should provide a valuable tool for a thorough molecular characterization of the enzyme.

Amino Acid Sequence↗
PubMed1997

Identification of essential aspartic acid and histidine residues of hormone-sensitive lipase: apparent residues of the catalytic triad.

It is expected that hormone-sensitive lipase (HSL), like most other lipases and esterases, adopts an alpha/beta-hydrolase fold and has a catalytic triad of serine, aspartic or glutamic acid, and histidine. Recently, we have published a three-dimensional model for the C-terminal catalytic domain of HSL, having an alpha/beta-hydrolase fold and with Ser-423(1), Asp-703 and His-733 in the catalytic triad (Contreras et al. (1996) J. Biol. Chem. 271, 31426-31430). It has been shown that Ser-423, situated in the motif GXSXG, is essential for catalysis (Holm et al. (1994) FEBS Lett. 344, 234-238). The suggested aspartic acid and histidine were here probed by site-directed mutagenesis. Mutants of residues Asp-703 and His-733 are devoid of both lipase and esterase activity, which is not the case for mutants of other tested aspartic acid and histidine residues. Thus, the presented data support the three-dimensional model structure with Asp-703 and His-733 as part of the traid.

Animals↗
PubMed1996

Hormone-sensitive lipase is structurally related to acetylcholinesterase, bile salt-stimulated lipase, and several fungal lipases. Building of a three-dimensional model for the catalytic domain of hormone-sensitive lipase.

Hormone-sensitive lipase is the key enzyme in the mobilization of fatty acids from adipose tissue, thereby playing a crucial role in the overall energy homeostasis in mammals. Its activity is stimulated by catecholamines through cAMP-dependent phosphorylation of a single serine, a process that is prevented by insulin. This regulatory property is unique to this enzyme among all known lipases and has been acquired during evolution through insertion of a regulatory module into an ancestral lipase. Sequence alignments have failed to detect significant homology between hormone-sensitive lipase and the rest of the mammalian lipases and esterases, to which this enzyme is only very distantly related. In the present work, we report the finding of a remarkable secondary structure homology between hormone-sensitive lipase and the enzymes from a superfamily of esterases and lipases that includes acetylcholinesterase, bile salt-stimulated lipase, and several fungal lipases. This finding, based on the identification of the secondary structure elements in the hormone-sensitive lipase sequence, has allowed us to construct a three-dimensional model for the catalytic domain of hormone-sensitive lipase. The model reveals the topological organization, predicts the components of the catalytic triad, suggests a three-dimensional localization of the regulatory module, and provides a valuable tool for the future study of structural and functional aspects of this metabolically important enzyme.

Acetylcholinesterase↗
PubMed1996

Domain-structure analysis of recombinant rat hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.

Amino Acid Sequence↗
PubMed1994

Identification of the active site serine of hormone-sensitive lipase by site-directed mutagenesis.

The consensus pentapeptide GXSXG is found in virtually all lipases/esterases and generally contains the active site serine. The primary sequence of hormone-sensitive lipase contains a single copy of this pentapeptide, surrounding Ser-423. We have analyzed the catalytic role of Ser-423 by site-directed mutagenesis and expression of the mutant hormone-sensitive lipase in COS cells. Substitution of Ser-423 by several different amino acids resulted in the complete abolition of both lipase and esterase activity, whereas mutation of other conserved serine residues had no effect on the catalytic activity. These results strongly suggest that Ser-423 is the active site serine of hormone-sensitive lipase.

Amino Acid Sequence↗
PubMed1994

Hormone-sensitive lipase: structure, function, evolution and overproduction in insect cells using the baculovirus expression system.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in the hydrolysis of stored triacylglycerols and is thereby a key enzyme in lipid metabolism and overall energy homeostasis. The gene organization of human HSL indicates that each putative functional region is encoded by a different exon, raising the possibility that HSL is a mosaic protein. The catalytic serine (Ser423), as shown by site-directed mutagenesis, is encoded by exon 6. The phosphorylation site for cAMP-mediated activity control and a second site, which is presumably phosphorylated by 5' AMP-activated kinase, are encoded by exon 8, and a putative lipid-binding region is encoded by the ninth and last exon. Besides the catalytic site serine motif (GXSXG), found in virtually all lipases, a sequence similarity between the region surrounding the catalytic site of HSL and that of five prokaryotic enzymes has been found, but the functional basis of this is not yet understood. To resolve the 3-D structure of HSL, an expression system utilizing recombinant baculovirus and insect cells has been established. The expressed protein, 80 mg/l culture, has been purified to homogeneity and a partial characterization indicates that it has the same properties as HSL purified from rat adipose tissue.

Animals↗