Neuronal ceroid lipofuscinoses (NCL) represent a group of common progressive encephalopathies of children which have a global incidence of 1 in 12,500. These severe brain diseases are divided into three autosomal recessive subtypes, assigned to different chromosomal loci. The infantile subtype of NCL (INCL), linked to chromosome 1p32, is characterized by early visual loss and rapidly progressing mental deterioration, resulting in a flat electroencephalogram by 3 years of age; death occurs at 8 to 11 years, and characteristic storage bodies are found in brain and other tissues at autopsy. The molecular pathogenesis underlying the selective loss of neurons of neocortical origin has remained unknown. Here we report the identification, by positional candidate methods, of defects in the palmitoyl-protein thioesterase gene in all 42 Finnish INCL patients and several non-Finnish patients. The most common mutation results in intracellular accumulation of the polypeptide and undetectable enzyme activity in the brain of patients.

PPT1 and PPT2 encode two lysosomal thioesterases that catalyze the hydrolysis of long chain fatty acyl CoAs. In addition to this function, PPT1 (palmitoyl-protein thioesterase 1) hydrolyzes fatty acids from modified cysteine residues in proteins that are undergoing degradation in the lysosome. PPT1 deficiency in humans causes a neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (also known as infantile Batten disease). In the current work, we engineered disruptions in the PPT1 and PPT2 genes to create ''knockout'' mice that were deficient in either enzyme. Both lines of mice were viable and fertile. However, both lines developed spasticity (a ''clasping'' phenotype) at a median age of 21 wk and 29 wk, respectively. Motor abnormalities progressed in the PPT1 knockout mice, leading to death by 10 mo of age. In contrast, the majority of PPT2 mice were alive at 12 mo. Myoclonic jerking and seizures were prominent in the PPT1 mice. Autofluorescent storage material was striking throughout the brains of both strains of mice. Neuronal loss and apoptosis were particularly prominent in PPT1-deficient brains. These studies provide a mouse model for infantile neuronal ceroid lipofuscinosis and further suggest that PPT2 serves a role in the brain that is not carried out by PPT1.

No abstract

Background:The substrates and regulation of DHHC protein palmitoyl acyltransferases (PATs) are largely unknown. Results: Flotillin-2 palmitoylation is abolished in DHHC5 gene-targeted neural stem cells, and neuronal differentiation induces DHHC5 turnover. Conclusion: Flotillin-2 is a substrate for DHHC5, which is regulated at the protein level. Significance: The paper describes an approach to PAT substrate identification and a new PAT regulation mechanism.

Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.

Palmitoyl-protein thioesterase is a newly described long chain fatty-acid hydrolase that removes fatty acyl groups from modified cysteines in proteins. We have recently identified palmitoyl-protein thioesterase as the defective enzyme in the recessive hereditary neurological degenerative disorder infantile neuronal ceroid lipofuscinosis (Vesa, J., Hellsten, E., Verkruyse, L. A., Camp, L. A. , Rapola, J., Santavuori, P., Hofmann, S. L., and Peltonen, L. (1995) Nature 376, 584-587). A defect in a lysosomal enzyme had been postulated for the disease, but until recently, the relevant defective lysosomal enzyme had not been identified. In this paper, we present evidence for the lysosomal localization of palmitoyl-protein thioesterase. We show that COS cells take up exogenously supplied palmitoyl-protein thioesterase intracellularly and that the cellular uptake is blocked by mannose 6-phosphate, a hallmark of lysosomal enzyme trafficking. The enzyme contains endoglycosidase H-sensitive oligosaccharides that contain phosphate groups. Furthermore, palmitoyl-protein thioesterase cosediments with lysosomal enzyme markers by Percoll density gradient centrifugation. Interestingly, the pH optimum for the enzyme is in the neutral range, a property shared by two other lysosomal enzymes that remove post-translational protein modifications. These findings suggest that palmitoyl-protein thioesterase is a lysosomal enzyme and that infantile neuronal ceroid lipofuscinosis is properly classified as a lysosomal storage disorder.

Mutations in palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme that removes fatty acyl groups from cysteine residues in modified proteins, cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis. The accumulation of undigested substrates leads to the formation of neuronal storage bodies that are associated with the clinical symptoms. Less severe forms of PPT1 deficiency have been found recently that are caused by a distinct set of PPT1 mutations, some of which retain a small amount of thioesterase activity. We have determined the crystal structure of PPT1 with and without bound palmitate by using multiwavelength anomalous diffraction phasing. The structure reveals an ␣͞␤-hydrolase fold with a catalytic triad composed of Ser115-His289-Asp233 and provides insights into the structural basis for the phenotypes associated with PPT1 mutations.

Mutations in a newly described lysosomal enzyme, palmitoyl-protein thioesterase (PPT), were recently shown to be responsible for an autosomal recessive neurological disorder prevalent in Finland, infantile neuronal ceroid lipofuscinosis. The disease results in blindness, motor and cognitive deterioration, and seizures. Characteristic inclusion bodies (granular osmiophilic deposits [GROD]) are found in the brain and other tissues. The vast majority of Finnish cases are homozygous for a missense mutation (R122W) that severely affects PPT enzyme activity, and the clinical course in Finnish children is uniformly rapidly progressive and fatal.To define the clinical, biochemical, and molecular genetic characteristics of subjects with PPT deficiency in a broader population, we collected blood samples from U.S. and Canadian subjects representing 32 unrelated families with neuronal ceroid lipofuscinosis who had GROD documented morphologically. We measured PPT activity and screened the coding region of the PPT gene for mutations. In 29 of the families, PPT deficiency was found to be responsible for the neurodegenerative disorder, and mutations were identified in 57 out of 58 PPT alleles. One nonsense mutation (R151X) accounted for 40% of the alleles and was associated with severe disease in the homozygous state. A second mutation (T75P) accounted for 13% of the alleles and was associated with a late onset and protracted clinical course. A total of 19 different mutations were found, resulting in a broader spectrum of clinical presentations than previously seen in the Finnish population. Symptoms first appeared at ages ranging from 3 mo to 9 yr, and about half of the subjects have survived into the second or even third