Structure-function relationships of hormone-sensitive lipase

Sammanfattning: The primary role of hormone-sensitive lipase (HSL), an 84 kDa enzyme of 768 amino acids (in the rat protein), is to hydrolyse stored triacylglycerols in adipocytes of white adipose tissue. Catecholamines and insulin regulate lipolysis through cellular signalling. A major target of this regulation is HSL, activated through phosphorylation by cAMP-dependent protein kinase (cAMP-PK). Insulin activates a cAMP phosphodiesterase, leading to decreased cAMP levels and deactivation HSL. The structure-function relationships of HSL have been studied using the recombinant rat HSL expressed in COS cells and insect cells. The latter has provided large amounts of HSL protein that was purified to more than 95 % purity. The recombinant protein was shown to be identical to HSL purified from rat adipocytes with respect to different activities, specific activity and phosphorylation/activation by cAMP-PK. The primary structure of HSL from different species was compared to each other and to that of a bacterial lipase. Those parts that aligned to the bacterial lipase were suggested to form the a/b-hydrolase fold core of HSL, a fold common to lipases, esterases and other enzymes. A separate study confirmed this localisation and has provided a three-dimensional model for the catalytic core of HSL. A stretch of ca. 200 amino containing identified phosphorylation sites intervenes this fold and is thought to be a regulatory module. Primary-, secondary- and tertiary structure analysis provided suggestions for the amino acids of the catalytic triad (another common feature of lipases and esterases). All three suggested amino acids were probed by site-directed mutagenesis and found to be essential for activity. Other conserved amino acids, that were chosen as controls, were not important for activity. Thus, the catalytic triad of rat HSL is thought to be Ser-423, Asp-703 and His-733. The sequence analysis also suggested that HSL has at least two structural domains, one having the a/b-hydrolase fold core and regulatory module, and a second N-terminal domain of unknown function. HSL, subjected to denaturation and limited proteolysis, was monitored by measurements of activities, circular dichroism and fluorescence spectroscopy. These studies clearly support the concept that HSL has two structural domains. Cleavage peptides of HSL were identified by mass spectrometry after proteolysis and electrophoretic separation, and confirmed the location of predicted exposed regions, i.e. the regulatory module and a hinge region between the structural domains. Analysis of the hydropathic pattern of HSL, supported these observations, by revealing that hydrophilic regions correlate with the exposed regions. The N-terminal domain is mostly amphipathic whereas the a/b-hydrolase fold core is mostly hydrophobic. Thus, detergent/membrane interaction sites are thought to be located to the N-terminal domain and parts of the hydrolase core. The picture of HSL emerging from these studies is that HSL probably has two structural domains separated by a short hinge region. The N-terminal domain is composed of ca. the 320 first residues and may mediate interactions with the intracellular lipid droplet substrate. The C-terminal domain is composed of ca. the last 430 residues of which half constitutes the a/b-hydrolase fold core and harbours the catalytic triad, whereas the rest is suggested to be a regulatory module containing the phosphorylation sites.