Mutations In Palmitoyl-protein Thioesterase Research Articles

Mutations in palmitoyl-protein thioesterase 1 alter exocytosis and endocytosis at synapses in Drosophila larvae

Infantile-onset neuronal ceroid lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate group from its substrate proteins, which may include presynaptic proteins like SNAP-25, cysteine string protein (CSP), dynamin, and synaptotagmin. The fruit fly, Drosophila melanogaster, has been a powerful model system for studying the functions of these proteins and the molecular basis of neurological disorders like the NCLs. Genetic modifier screens and tracer uptake studies in Ppt1 mutant larval garland cells have suggested that Ppt1 plays a role in endocytic trafficking. We have extended this analysis to examine the involvement of Ppt1 in synaptic function at the Drosophila larval neuromuscular junction (NMJ). Mutations in Ppt1 genetically interact with temperature sensitive mutations in the Drosophila dynamin gene shibire, accelerating the paralytic behavior of shibire mutants at 27 °C. Electrophysiological work in NMJs of Ppt1-deficient larvae has revealed an increase in miniature excitatory junctional potentials (EJPs) and a significant depression of evoked EJPs in response to repetitive (10 hz) stimulation. Endocytosis was further examined in Ppt1-mutant larvae using FM1–43 uptake assays, demonstrating a significant decrease in FM1–43 uptake at the mutant NMJs. Finally, Ppt1-deficient and Ppt1 point mutant larvae display defects in locomotion that are consistent with alterations in synaptic function. Taken together, our genetic, cellular, and electrophysiological analyses suggest a direct role for Ppt1 in synaptic vesicle exo- and endocytosis at motor nerve terminals of the Drosophila NMJ.

Two novel mutations in palmitoyl-protein thioesterase gene in two Chinese babies with infantile neuronal ceroid lipofuscinosis

To search for possible novel mutations in palmitoyl-protein thioesterase 1 (PPT1) gene in two Chinese babies with infantile neuronal ceroid lipofuscinosis (INCL). Two probands with INCL, confirmed clinically and pathologically, were used for mutation search in PPT1 gene. Onset of the disease occurred before the age of 1 year and they mainly showed progressive mental and motor retardation. The 9 coding exons and their flanking intron sequences of palmitoyl-protein thioesterase 1 (PPT1) gene were amplified by using PCR and sequenced. The parents of proband 1 were also examined. One splicing mutation and two missense mutations were identified in the two probands: the proband 1 carrying a compound heterozygous mutation of a IVS1 + 1G-->A mutation in intron 1 and a c550G-->A mutation in exon 6 leading to the amino acid substitution of E184K. Additionally, the parents of the proband 1 also harbored one of the mutations of the patient, respectively. The proband 2 carrying a homozygous mutation of c272A-->C in exon 3, which resulted in the amino acid substitutions of Q91P. The IVS1 + 1G-->A mutation and Q91P mutation are novel mutations, which lead to INCL. The genetic abnormalities of PPT1 in Chinese patients may not be completely the same as those in the patients of other regions of the world.

Biochemical analysis of mutations in palmitoyl-protein thioesterase causing infantile and late-onset forms of neuronal ceroid lipofuscinosis.

Deficiency in a recently characterized lysosomal enzyme, palmitoyl-protein thioesterase (PPT), leads to a severe neurodegenerative disorder of children, infantile neuronal ceroid lipofuscinosis (NCL). Over 36 different mutations in the PPT gene have been described, and missense mutations have been interpreted in the light of the recently solved X-ray crystallographic structure of PPT. In the current study, we assessed the biochemical impact of mutations through the study of cells derived from patients and from the expression of recombinant PPT enzymes in COS and Sf9 cells. All missense mutations associated with infantile NCL showed no residual enzyme activity, whereas mutations associated with late-onset phenotypes showed up to 2.15% residual activity. Two mutations increased the K(m) of the enzyme for palmitoylated substrates and were located in positions that would distort the palmitate-binding pocket. An initiator methionine mutation (ATG-->ATA) in two late-onset patients was expressed at a significant level in COS cells, suggesting that the ATA codon may be utilized to a clinically important extent in vivo. The most common PPT nonsense mutation, R151X, was associated with an absence of PPT mRNA. Mannose 6-phosphate modification of wild-type and mutant PPT enzymes was grossly normal at the level of the phosphotransferase reaction. However, mutant PPT enzymes did not bind to mannose 6-phosphate receptors in a blotting assay. This observation was related to the failure of the mutant expressed enzymes to gain access to 'uncovering enzyme' (N-acetylglucosamine-1-phosphodiester alpha-N-acetyl glucosaminidase), presumably due to a block in transit out of the endoplasmic reticulum, where mutant enzymes are degraded.

The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis.

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 alpha/beta-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.

Detection of eight novel palmitoyl protein thioesterase (PPT) mutations underlying infantile neuronal ceroid lipofuscinosis (INCL;CLN1).

The infantile form of neuronal ceroid lipofuscinosis (INCL; CLN1) is the earliest onset form of the neuronal ceroid lipofuscinoses (NCL), a group of progressive encephalopathies of children. INCL is caused by mutations in the palmitoyl protein thioesterase (PPT) gene, and we report here eight novel INCL mutations in PPT. Five of the mutations, c.456C>A, c.162-163insA, c.174-175delG, c.774-775insA, and a splice acceptor mutation IVS1-2A>G in intron 1, caused the generation of a premature STOP codon either directly or after a frameshift. One mutation was a three-bp insertion in exon 2 (c. 132-133insTGT) leading to insertion of one extra cysteine (Ser44-insCys-Cys45), and another mutation, a 3-bp deletion in exon 3 (c.249-251delCTT), led to deletion of Phe84 in PPT. A splice acceptor mutation IVS6-1G>T in intron 6 can be predicted to cause skipping of exon 7 in PPT. All of these novel mutations were associated with the classical phenotype of INCL, with the first symptoms starting around 12 months of age. The severe phenotypes could be explained by the nature of the novel mutations: five are predicted to lead to premature translational termination, thus abolishing the active site of PPT, and three will probably cause a misfolding of the nascent polypeptide. Thirty-five percent (7/20) of the disease alleles in these 11 families contained the most prevalent c.451C>T missense mutation outside Finland [Das et al., 1998]. Consequently, 31 different mutations underlying INCL have been found so far, the majority leading to classical INCL.

Molecular genetics of the neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterised by the accumulation of autofluorescent storage material in neurons and other cell types. The clinical features include visual impairment, progressive myoclonic epilepsy, and cognitive decline reflecting progressive neurodegeneration. The NCLs are subdivided into several subtypes according to age of onset, clinical course, and ultrastructure of the storage material. The molecular genetic basis of this group of disorders has recently been clarified. Mutations in the gene encoding a lysosomal enzyme, palmitoyl protein thioesterase (PPT), cause infantile NCL (locus CLN1 on chromosome 1p32) or Haltia-Santavuori disease. This Finnish disease is characterised ultrastructurally by granular osmiophilic deposits (GRODs). Juvenile-onset NCL with GRODs also is caused by mutations in PPT. Classic late-infantile NCL (Jansky-Bielschowsky disease) is caused by mutations in a gene encoding a pepstatin-insensitive lysosomal peptidase (CLN2 on chromosome 11p15), and juvenile-onset NCL (Batten disease) is caused by mutations in a gene encoding a 438-amino-acid membrane protein (CLN3 on chromosome 16p12) of unknown function. A locus for Finnish variant late-infantile NCL, CLN5, has been mapped to chromosome 13q22 and a locus for variant late-infantile NCL, CLN6, to chromosome 15q21-23. These and further advances will allow the molecular basis of the NCLs to be elucidated and may lead to new strategies for diagnosis and treatment.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

CDNA and Genomic Cloning of Human Palmitoyl-Protein Thioesterase (PPT), the Enzyme Defective in Infantile Neuronal Ceroid Lipofuscinosis

Palmitoyl-protein thioesterase (PPT) is a small glycoprotein that removes palmitate groups from cysteine residues in lipid-modified proteins. We recently reported mutations in PPT in patients with infantile neuronal ceroid lipofuscinosis (INCL), a severe neurodegenerative disorder (J. Vesaet al.,1995,Nature376: 584–587). INCL is characterized by the accumulation of proteolipid storage material in brain and other tissues, suggesting that the disease is a consequence of abnormal catabolism of acylated proteins. In the current paper, we report the sequence of the human PPT cDNA and the structure of the human PPT gene. The cDNA predicts a protein of 306 amino acids that contains a 25-amino-acid signal peptide, three N-linked glycosylation sites, and consensus motifs characteristic of thioesterases. Northern analysis of a human tissue blot revealed ubiquitous expression of a single 2.5-kb mRNA, with highest expression in lung, brain, and heart. The human PPT gene spans 25 kb and is composed of seven coding exons and a large eighth exon, containing the entire 3′-untranslated region of 1388 bp. AnAlurepeat and promoter elements corresponding to putative binding sites for several general transcription factors were identified in the 1060 nucleotides upstream of the transcription start site. The human PPT cDNA sequence and gene structure will provide the means for the identification of further causative mutations in INCL and facilitate genetic screening in selected high-risk populations.

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

The marine natural product didemnin B, currently in clinical trials as an antitumor agent, has several potent biological activities apparently mediated by distinct mechanisms. Our initial investigation of didemnin B resulted in the discovery of its GTP-dependent binding of the translation elongation factor EF1 alpha. This finding is consistent with the protein synthesis inhibitory activity of didemnin B observed at intermediate concentrations. To begin to dissect the mechanisms involved in the cytostatic and immunosuppressive activities of didemnin B, observed at low concentrations, additional didemnin-binding proteins were sought. Here we report the purification of a 36-kDa glycosylated didemnin-binding protein from bovine brain lysate. Cloning of the human cDNA encoding this protein revealed a strong sequence similarity with palmitoyl protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and the G alpha s subunits of heterotrimeric GTP-binding proteins in vitro. Mutations in PPT have recently been shown to be responsible for infantile neuronal ceroid lipofuscinosis, which is a severe brain disorder characterized by progressive loss of brain function and early death.