Hormone-sensitive lipase: Molecular analyses of the human gene; structural and evolutionary aspects on expression, alternative splicing and cold adaptation

Sammanfattning: Hormone-sensitive lipase (HSL) is a key enzyme in adipose tissue that hydrolyses stored fat (triacylglycerol). The activity of HSL is under strict hormonal and neural control. Lipolytic agents, such as catecholamines, increase the cAMP levels, resulting in an activation of cAMP-dependent protein kinase (PKA), which in turn phosphorylates and activates HSL. This thesis describes the structural organisation of the human gene, the human and garden dormouse HSL cDNA sequence and the primary characterisation of the promoter directing HSL expression in human white adipose tissue. The HSL locus, positioned on the long arm of chromosome 19 (19q13.13), was shown not to be duplicated to the short arm, as previously suggested. The human HSL gene consists of nine coding exons (exons 1 to 9) spanning 11 kb, which are common for all human HSL transcripts studied to date. Moreover, there are four known distinct 5' exons (T1, T2, A and B, located 16, 12,5, 13, and 1,5 kb upstream exon 1), which are used in a mutually exclusive manner. The expression of the HSL gene is regulated in a complex tissue- and, most likely, species-specific manner. In human testis mRNA both T1 and T2 are present, whereas in human white adipose tissue HSL expression is directed from the promoter upstream of exon B. Both exon A and B are found in HSL mRNA from white adipocytes in garden dormouse (Eliomys quercinus sp.), indicating a species difference compared to human adipocytes. Curiously, garden dormouse HSL is more similar to human than to rat and mouse HSL. Phylogenetic analyses of all four sequences support this relationship, suggesting paraphylism among rodents. Up to 40% of human HSL lacks exon 6, due to a species-specific, in-frame exon skipping during the pre-mRNA splicing. The skipping occurs because of species differences in the sequences within and just downstream exon 6. The truncated mRNA is translated and the protein product (HSL-S) is, as expected, devoid of both lipase and esterase activities, due to lack of the catalytic site serine. However, the phosphorylation sites, encoded by exon 8, appear to be exposed, since HSL-S acts as substrate for PKA. HSL displays a high catalytic activity at low temperatures. A loop structure located close to the active site contains a stack of glycine residues. The contribution of these residues to the cold adaptation property of HSL was investigated through site-directed mutagenesis. It was concluded that the multiple glycine residues in this loop are important for enzyme function but are not alone responsible for rendering HSL cold adapted.