Characterization of Drosophila palmitoyl-protein thioesterase 1

Abstract

Batten disease or neuronal ceroid lipofuscinoses (NCL) are a group of genetic neurodegenerative diseases that primarily afflict infants and children and are characterized by progressive loss of brain functions caused by the death of central nervous system (CNS) neurons. The most severe form of the disease is infantile NCL (INCL). INCL is caused by mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene, which encodes a palmitoyl-protein thioesterase 1 enzyme that cleaves long-chain fatty acids from S-acylated proteins within the lysosome. How the loss of this activity causes the death of CNS neurons is not known. A PPT1 homolog and palmitoyl-protein thioesterase 1 enzyme activity were characterized in Drosophila melanogaster as an initial step in developing Drosophila as a model system for studying the etiology of INCL. Predicted gene CG12108 in region 8A2 of the X chromosome is 55% identical and 72% similar to human PPT1 and contains conserved catalytic residues and sites of glycosylation. Northern-blot hybridizations revealed a major 1.5 kb CG12108 transcript in unfertilized eggs, embryos, larvae, pupae, adult head and thorax, ovary, testis, and S2 tissue culture cells, as well as several minor mRNA species in some tissues. Levels of the 1.5 kb transcript were fairly uniform among tissues except in testis, where the transcript was enriched 5-fold. The same tissues also contained palmitoyl-protein thioesterase 1 enzyme activity measured using the fluorometric substrate 4-methylumbelliferyl-6-thiopalmitoyl-β-d-glucoside. Enzyme activity was highest in testis and varied among the other tissues to a greater extent than did CG12108 message, suggesting that CG12108 is subjected to post-transcriptional regulation. Finally, flies homozygous for a deletion that removes CG12108 and three unrelated neighboring genes had less than 3% of wildtype levels of enzyme activity, consistent with CG12108 encoding functional palmitoyl-protein thioesterase 1 activity and being the fly ortholog of human PPT1. CG12108 has been appropriately renamed Ppt1.

Introduction

Batten disease, or neuronal ceroid lipofuscinoses (NCL), consists of a group of genetic neurodegenerative diseases that primarily afflict infants and children (see Mitchison and Mole, 2002, Wisniewski and Zhong, 2001 for overviews). The diseases are all characterized by a progressive loss of brain functions caused by the dysfunction and eventual death of CNS neurons. Collectively, NCLs are the most common neurogenetic diseases of childhood, with an incidence that varies from 0.1 to 7 per 100,000 (discussed in Wisniewski et al., 2001). There are eight forms of the disease recognized clinically: infantile (INCL), late infantile (LINCL), juvenile (JNCL), adult (ANCL), Finnish LINCL, Gypsy/Indian LINCL, Turkish LINCL, and progressive epilepsy with mental retardation. The same diseases are referred to genetically as CLN1 through CLN8, respectively, and are transmitted predominantly as autosomal recessive mutations. The characteristic that defines all forms of Batten disease as a distinct and related group is the accumulation of autofluorescent lipopigment inclusions in many cell types of the body, including neurons (see Wisniewski et al., 2001 for overview). Inclusions are typically located in lysosomes; thus, NCLs are often categorized as lysosomal storage diseases. The predominant localization of lipopigment inclusions within lysosomes, combined with recent biochemical and genetic evidence, supports the conclusion that all forms of Batten disease are caused by mutations that either directly or indirectly disrupt normal lysosome function. Defective lysosomal function, in turn, causes accumulation of lipopigment inclusions, neuron dysfunction, and eventually cell death. Consistent with this hypothesis, three of the five identified CLN genes encode proteins that localize to lysosomes. CLN1, mutations to which cause INCL, encodes a lysosomal palmitoyl-protein thioesterase (Vesa et al., 1995 and see below). CLN2, mutations to which cause LINCL, encodes a lysosomal peptidase (Sleat et al., 1997), and CLN3, mutations to which cause JNCL, encodes a lysosomal membrane protein of unknown function (Jarvela et al., 1998). Rapid progress is being made in understanding the causes of Batten Disease, stimulated in large part by the identification of CLN genes.

Of the eight forms of Batten disease, INCL is most severe. It has the earliest age of onset, 6–18 months, and the most rapid rate of neurodegeneration, with premature death occurring between years 8 and 13 (see Hofmann et al., 2001 for overview of INCL). INCL is caused by mutations in the palmitoyl-protein thioesterase 1 gene (PPT1) (Vesa et al., 1995). PPT1 encodes a soluble lysosomal enzyme that hydrolyzes the thioester bond that attaches long-chain fatty acids, mostly palmitate, to the cysteine residue of S-acylated proteins (Camp and Hofmann, 1993, Camp et al., 1994). PPT1 is expressed throughout the body and likely provides a general housekeeping function, although the normal in vivo lipoprotein substrates of PPT1 are not known (Salonen et al., 1998, Schriner et al., 1996, Suopanki et al., 1999). Recent studies also suggest possible roles for PPT1 outside the lysosome, including roles in synaptosome function and in the signaling pathways that regulate apoptosis (Ahtiainen et al., 2003, Cho and Dawson, 2000, Cho et al., 2000, Lehtovirta et al., 2001). Disease severity is inversely correlated with PPT1 enzyme activity such that null mutations are invariably associated with severe infantile NCL, while some hypomorphic alleles are associated with late infantile and juvenile onset phenotypes (summarized in Hofmann et al., 2001). It is now estimated that PPT1 mutations account for 20–25% of all NCLs in the United States (Hofmann et al., 2001). Identification of PPT1 as the CLN1 gene was a significant advance and has stimulated a rapid expansion in our knowledge of INCL, including the recent development of a murine model for the disease (Gupta et al., 2001). Despite these advances, our understanding of the etiology of Batten disease at a molecular level is still limited, both in terms of our general understanding of lysosome biology as it relates to accumulation of lipopigment inclusions, and more specifically, the relationship between loss of PPT1 enzyme activity and neuron cell death.

Over the last hundred years, studies of the fruitfly, Drosophila melanogaster, have contributed significantly to our understanding of many basic biological processes. In recent years, sequencing of the Drosophila genome, combined with reverse genetic studies, has demonstrated the utility of using Drosophila to study human genetic diseases, including neurological diseases of the brain. Of 287 known human disease genes, 62% have direct homologues in Drosophila; of these, 38 of 59 neurological disease genes are represented (Adams et al., 2000, Fortini et al., 2000, Rubin et al., 2000). Although flies and humans are phylogenetically divergent, basic cellular and molecular features of numerous disease manifestations are conserved. Drosophila has proven its value in helping to unravel mechanisms underlying a number of complex human neurological diseases including Huntington's, Parkinson's, Machado-Joseph Disease, and Spinocerebellar Ataxia type 1 (Feany and Bender, 2000, Fernandez-Funez et al., 2000, Jackson et al., 1998, Warrick et al., 1998). We report here the characterization of a PPT1 homolog and palmitoyl-protein thioesterase 1 enzyme activity in Drosophila as an initial step in evaluating Drosophila as a potential model for studying the etiology of INCL.