Lipases and LCAT

From LipidomicsWiki

Recent scientific advances have revealed the identity of several enzymes involved in the synthesis, storage and catabolism of intracellular neutral lipid storage droplets. An enzyme
that hydrolyzes stored triacylglycerol (TG), triacylglycerol hydrolase (TGH), was purified from porcine, human and murine liver microsomes. In rodents, TGH is highly expressed in liver as well as heart, kidney, small intestine and adipose tissues, while in humans TGH is mainly expressed in the liver, adipose and small intestine. TGH localizes to the endoplasmic
reticulum and lipid droplets. The TGH genes are located within a cluster of carboxylesterase genes on human and mouse chromosomes 16 and 8, respectively. TGH hydrolyzes stored
TG, and in the liver, the lipolytic products are made available for VLDL-TG synthesis. Inhibition of TGH activity also inhibits TG and apolipoprotein B secretion by primary hepatocytes. A role for TGH in basal TG lipolysis in adipocytes has been proposed. TGH expression and activity is both developmentally and hormonally regulated. A model for the function of TGH is presented and discussed with respect to tissue specific functions (Dolinsky et al. 2004). The murine TGH promoter sequence (Douglas et al. 2001) shares 59% identity to orthologous rat (Nataranjan et al. 1998) and 46% identity to the human (Langmann et al. 1997) TGH promoters. All three promoters share several common binding sites for transcription factors, suggesting that the orthologous TGH genes have evolutionarily conserved transcriptional regulatory patterns (fig. 89). A TATA box does not precede the
transcription start site. Potential binding sites for transcription factors include three Sp1 binding sites, a NF-1, a peroxisomal proliferator-activated receptor response element and
three sterol response element-like sequences.

Various studies observed increased expression of rat and mouse TGH mRNA and protein in the liver at the time of weaning, coincident with enhanced ability to secrete VLDL (Lehner et al. 1999; Morgan et al. 1994; Douglas et al. 2001; Trickett et al. 2001). This age-dependent expression appeared to be related to dietary changes at the time of weaning and
independent of hepatic differentiation, since TGH expression was unchanged in regenerating liver that undergoes dedifferentiation and acquires fetal and neonatal features following
partial hepatectomy (Lehner et al. 1999). The murine TGH promoter was utilized to determine whether the increased expression of TGH seen at the time of weaning was linked
to transcriptional regulation and to identify potential transcription factors and cis-acting DNA elements that might mediate the observed developmental expression of TGH in liver.
Electrophoretic mobility shift assays demonstrated enhanced binding to the murine TGH promoter of hepatic nuclear proteins from 27-day-old weaned mice compared with 7-day-old
suckling mice (Douglas et al. 2001). DNase I footprint analysis localized binding of nuclear proteins to two regions within the promoter: site A, which contains a Sp1 binding site, and
site B, which contains a degenerate E-box (Douglas et al. 2001). Competitive electromobility shift and supershift assays demonstrated that site A binds Sp1 and Sp3 transcription factors and transcriptional activation assays in Schneider SL-2 insect cells demonstrated that Sp1 was a potent activator of the TGH promoter (Douglas et al. 2001). Sp1 is a ubiquitous nuclear protein that activates the transcription of a wide variety of genes (Suske 1999).

TATA-less promoters have been shown to be particularly sensitive to regulation by the Sp family of proteins (Dynan and Tjian 1983; Anderson and Freytag 1991). Alignment of the 5¢
proximal promoters of the murine, rat and human TGH genes demonstrated that regions termed A and B sequences are evolutionarily conserved (Douglas et al. 2001). Indeed,
others have observed that reporter constructs containing the conserved site A sequence activate transcription, while their elimination reduces promoter activity (Langmann et al.
1997; Nataranjan et al. 1998). A role for Sp1 in the phorbol ester-induced differentiationdependent expression of human TGH in the macrophage THP-1 cell line has been
demonstrated (Langmann et al. 1997). The transcription factor(s) that mediates the induction of murine TGH expression during 3T3-L1 adipocyte differentiation are not known (Dolinsky et al. 2003), but are the subject of current investigation in our laboratory. Since TGH expression occurs at a late stage of adipocyte differentiation, clearly peroxisomal proliferator-activated receptor (PPAR)g is potentially involved in the induction of TGH expression in mature 3T3-L1 adipocytes. We explored this possibility using a PPARg agonist that exhibited 1000-fold selectivity over the other receptor isoforms (Oliver et al. 2001). Though TGH expression was increased when the PPARg agonist was present throughout the differentiation process, a direct effect of PPARg on TGH expression was observed (Dolinsky et al. 2003). However, it is unclear whether the cholesterol mediated regulation of TGH expression is due
to SREBPs, oxysterol nuclear receptors or an indirect mechanism. TGH is subject to regulation by glucocorticoids in the mouse (Dolinsky et al. 2004). Glucocorticoids cause an
increase in circulating TG and increased TG synthesis and storage (Glenny and Brindley 1978; Krausz et al. 1981; Cole et al. 1982; Staels et al. 1991). Dexamethasone (Dex) is a
potent analog of glucocorticoids (cortisol and corticosterone) and induces similar effects as natural glucocorticoids (Staels et al. 1991). TGH mRNA, protein expression and hepatic
microsomal esterase activity were decreased by Dex, whereas hepatic microsomal DGAT activity and DGAT-1 and -2 expression were increased (Dolinsky et al. 2004). This result
correlates with decreased hepatic esterase activity (Morgan et al. 1994; Hosokawa et al. 1993; Zhu et al. 2000).

Two consensus sites for N-linked glycosylation have been identified at Asn79 and 489 in murine TGH and one at Asn79 in the human. Glycan detection assay of the human TGH
indicates that the mature TGH protein was glycosylated (Alam et al. 2002). However, mutation of Asn79 to Ala or Gln did not dramatically reduce human TGH activity, indicating
that glycosylation was not necessary for esterase activity (Alam et al. 2002; D. Gilham et al., unpublished). The primary sequence of human TGH contains a putative Tyr phosphorylation site at Tyr118, as well as seven potential Ser/Thr consensus sequences for phosphorylation by casein kinase II (CKII). The Tyr118 site is adjacent to the lid domain formed by the loop created by the disulfide bridge between Cys87 and Cys116, potentially regulating the opening of the lid and entry of substrate into the catalytic cleft and the active site. Regulation of enzyme activity via this mechanism is an area of active study in our laboratory. Pertinent to TGH, several CKII target proteins such as the bile salt-dependent lipase are present in the ER lumen (Verine et al. 2001). Reversible phosphorylation of rat TGH at Ser506 has been reported to increase CE hydrolase activity in isolated cytosolic fraction by 100–140% [70]. This mechanism of increased CE hydrolase activity in response to hormones associated with the fasted state (e.g. glucagon) is reminiscent of mobilization of TG and CE stores from adipose tissue by HSL. Phosphorylation of Ser506 is predicted to induce a conformational change that enhances binding of hydrophobic substrates, though the relevance of the regulation of rat TGH by phosphorylation is questionable, since Ser506 is not present in orthologous TGH amino acid sequences.