Jui Yun Lu

Molecular genetics of palmitoyl-protein thioesterase deficiency in the U.S.

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 decades of life.

Thematic review series: Lipid Posttranslational Modifications. Lysosomal metabolism of lipid-modified proteins

Much is now understood concerning the synthesis of prenylated and palmitoylated proteins, but what is known of their metabolic fate? This review details metabolic pathways for the lysosomal degradation of S-fatty acylated and prenylated proteins. Central to these pathways are two lysosomal enzymes, palmitoyl-protein thioesterase (PPT1) and prenylcysteine lyase (PCL). PPT1 is a soluble lipase that cleaves fatty acids from cysteine residues in proteins during lysosomal protein degradation. Notably, deficiency in the enzyme causes a neurodegenerative lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis. PCL is a membrane-associated flavin-containing lysosomal monooxygenase that metabolizes prenylcysteine to prenyl aldehyde through a completely novel mechanism. The eventual metabolic fates of other lipidated proteins (such as glycosylphosphatidylinositol-anchored and N-myristoylated proteins) are poorly understood, suggesting directions for future research.

Neuronal Ceroid Lipofuscinoses Caused by Defects in Soluble Lysosomal Enzymes (CLN1 and CLN2)

Infantile and classical late infantile neuronal ceroid lipofuscinoses (NCL) are two recent additions to the expanding spectrum of lysosomal storage disorders caused by deficiencies in lysosomal hydrolases. They are latecomers to the lysosomal storage disorders, probably because of the heterogeneous nature of the storage material, which precluded meaningful biochemical analysis. Infantile NCL is caused by deficiency in palmitoyl-protein thioesterase, an enzyme that hydrolyzes fatty acids from cysteine residues in lipid-modified proteins. Classical late-infantile NCL is caused by a deficiency in tripeptidyl amino peptidase-I, a lysosomal peptidase that removes three amino acids from the free amino terminus of peptides or small proteins. Late-onset forms of these disorders have been described. The clinical, biochemical, and molecular genetic aspects of these two latest lysosomal storage disorders are discussed in this review. In addition, approaches to treatment and future directions for research are examined.

Massive palmitoylation-dependent endocytosis during reoxygenation of anoxic cardiac muscle

In fibroblasts, large Ca transients activate massive endocytosis (MEND) that involves membrane protein palmitoylation subsequent to mitochondrial permeability transition pore (PTP) openings. Here, we characterize this pathway in cardiac muscle. Myocytes with increased expression of the acyl transferase, DHHC5, have decreased Na/K pump activity. In DHHC5-deficient myocytes, Na/K pump activity and surface area/volume ratios are increased, the palmitoylated regulatory protein, phospholemman (PLM), and the cardiac Na/Ca exchanger (NCX1) show greater surface membrane localization, and MEND is inhibited in four protocols. Both electrical and optical methods demonstrate that PTP-dependent MEND occurs during reoxygenation of anoxic hearts. Post-anoxia MEND is ablated in DHHC5-deficient hearts, inhibited by cyclosporine A (CsA) and adenosine, promoted by staurosporine (STS), reduced in hearts lacking PLM, and correlates with impaired post-anoxia contractile function. Thus, the MEND pathway appears to be deleterious in severe oxidative stress but may constitutively contribute to cardiac sarcolemma turnover in dependence on metabolic stress. DOI: http://dx.doi.org/10.7554/eLife.01295.001

Human recombinant palmitoyl-protein thioesterase-1 (PPT1) for preclinical evaluation of enzyme replacement therapy for infantile neuronal ceroid lipofuscinosis

Infantile neuronal ceroid lipofuscinosis (INCL, also known as Haltia-Santavuori disease) is a lysosomal storage disorder of infants and children characterized by blindness, seizures and a progressive neurodegenerative course. Recent clinical trials have involved neural stem cells and gene therapy directed to the central nervous system; however, enzyme replacement therapy has never been addressed. In the current paper, we describe the production of human recombinant PPT1 (the defective enzyme in INCL) by standard methods in Chinese Hamster Ovary (CHO) cells. The enzyme is largely mannose 6-phosphorylated as assessed by mannose 6-phosphate receptor binding (80% bound) and taken up rapidly by immortalized patient lymphoblasts, where clearance of PPT substrates was demonstrated (EC(50) of 0.25 nM after overnight incubation). When injected intravenously into PPT1-deficient mice, the clearance of recombinant human PPT1 from plasma was rapid, with a half-life of 10 min. Most of the injected dose was distributed to the kidney and liver and potentially corrective levels were also observed in heart, lung and spleen. Brain uptake was minimal, as expected based on experience with other intravenously administered lysosomal enzymes. The enzyme may be useful as an adjunct to central nervous system-directed therapies and could be used as a starting point for modifications designed to improve brain delivery.

Gene expression profiling in a mouse model of infantile neuronal ceroid lipofuscinosis reveals upregulation of immediate early genes and mediators of the inflammatory response

BackgroundThe infantile form of neuronal ceroid lipofuscinosis (also known as infantile Batten disease) is caused by hereditary deficiency of a lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1), and is characterized by severe cortical degeneration with blindness and cognitive and motor dysfunction. The PPT1-deficient knockout mouse recapitulates the key features of the disorder, including seizures and death by 7–9 months of age. In the current study, we compared gene expression profiles of whole brain from PPT1 knockout and normal mice at 3, 5 and 8 months of age to identify temporal changes in molecular pathways implicated in disease pathogenesis.ResultsA total of 267 genes were significantly (approximately 2-fold) up- or downregulated over the course of the disease. Immediate early genes (Arc, Cyr61, c-fos, jun-b, btg2, NR4A1) were among the first genes upregulated during the presymptomatic period whereas immune response genes dominated at later time points. Chemokine ligands and protease inhibitors were among the most transcriptionally responsive genes. Neuronal survival factors (IGF-1 and CNTF) and a negative regulator of neuronal apoptosis (DAP kinase-1) were upregulated late in the course of the disease. Few genes were downregulated; these included the α2 subunit of the GABA-A receptor, a component of cortical and hippocampal neurons, and Hes5, a transcription factor important in neuronal differentiation.ConclusionA molecular description of gene expression changes occurring in the brain throughout the course of neuronal ceroid lipofuscinosis suggests distinct phases of disease progression, provides clues to potential markers of disease activity, and points to new targets for therapy.

Intravenous high-dose enzyme replacement therapy with recombinant palmitoyl-protein thioesterase reduces visceral lysosomal storage and modestly prolongs survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis

PPT1-related neuronal ceroid lipofuscinosis (NCL) is a lysosomal storage disorder caused by deficiency in a soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1). Enzyme replacement therapy (ERT) has not been previously examined in a preclinical animal model. Homozygous PPT1 knockout mice reproduce the known features of the disease, developing signs of motor dysfunction at 5 months of age and death by around 8 months. In the current study, PPT1 knockout mice were treated with purified recombinant PPT1 (0.3 mg, corresponding to 12 mg/kg or 180 U/kg for a 25 g mouse) administered intravenously weekly either 1) from birth; or 2) beginning at 8 weeks of age. The treatment was surprisingly well tolerated and neither anaphylaxis nor antibody formation was observed. In mice treated from birth, survival increased from 236 to 271 days (p<0.001) and the onset of motor deterioration was similarly delayed. In mice treated beginning at 8 weeks, no increases in survival or motor performance were seen. An improvement in neuropathology in the thalamus was seen at 3 months in mice treated from birth, and although this improvement persisted it was attenuated by 7 months. Outside the central nervous system, substantial clearance of autofluorescent storage material in many tissues was observed. Macrophages in spleen, liver and intestine were especially markedly improved, as were acinar cells of the pancreas and tubular cells of the kidney. These findings suggest that ERT may be an option for addressing visceral storage as part of a comprehensive approach to PPT1-related NCL, but more effective delivery methods to target the brain are needed.

The effects of lysosomotropic agents on normal and INCL cells provide further evidence for the lysosomal nature of palmitoyl-protein thioesterase function

Fatty acylation of proteins on cysteine residues is a common post-translational modification that plays roles in protein-membrane and protein-protein interactions. Recently, we described a lysosomal palmitoyl-protein thioesterase that removes long-chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in palmitoyl-protein thioesterase results in a human lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis (INCL), which primarily affects the central nervous system. The pathological hallmark of the disorder is the accumulation of granular osmiophilic deposits (GROD) that resemble lipofuscin, or aging pigment. In previous work, we have shown that [35S]cysteine-labeled lipid thioesters derived from fatty acylated proteins accumulate in cultured cells derived from palmitoyl-protein thioesterase-deficient patients. In the present work, we show that the lipid cysteine thioesters accumulate in the lysosomal fraction, and we further show that the appearance of these compounds in the organic phase is blocked by inhibitors of lysosomal proteolysis, demonstrating through biochemical means the lysosomal nature of the site of palmitoyl-protein thioesterase action. Furthermore, substrates for palmitoyl-protein thioesterase accumulate even in normal cells after leupeptin or chloroquine treatment. This was demonstrated by subjecting extracts of treated cells to exhaustive proteolysis to release protein-bound cysteine lipid for analysis. Cysteamine, a lysosomotropic drug recently proposed for the treatment of INCL, was found to have effects similar to leupeptin and chloroquine, suggesting that its mechanism of action may be more complex than previously understood.

3 Positional candidate gene cloning of CLN1

Abstract Mutations in the CLN1 gene encoding palmitoyl-protein thiosterase (PPT) underlie the recessive neurodegenerative disorder, infantile Batten disease, or infantile neuronal ceroid lipofuscinosis (INCL). The CLN1 gene was mapped to chromosome 1p32 in the vicinity of a microsatellite marker HY-TM1 in a cohort of Finnish INCL families, and mapping of the PPT gene to the CLN1 critical region (and the discovery of mutations in PPT in several unrelated families) led to conclusive identification of PPT as the disease gene. PPT is a lysosomal thioesterase that removes fatty acids from fatty-acylated cysteine residues in proteins. The accumulation of fatty acyl cysteine thioesters can be reverse in INCL cells by the exogenous administration of recombinant PPT, which enters the cells through the mannose 6-phosphate receptor pathway. Over two dozen PPT mutations have been found in PPT-deficient patients worldwide. In the United States, all PPT-deficient patients show “GROD” histology but the age of onset of symptoms is later in some children due to the presence of missense mutations that result in enzymes with residual PPT activity. Now that INCL is known to be caused by a defect in a soluble lysosomal enzyme, appropriate therapies may be forthcoming. Prospects for therapy include enzyme replacement, stem cell transplantation gene therapy and metabolic therapy aimed at depleting the abnormal substrate accumulation in the disease.

Bacterial cytochromes P450: isolation and identification.

Publisher Summary This chapter discusses the isolation and identification bacterial cytochromes P450. The role of the members of the superfamily of proteins called cytochrome P450 in the oxidation of organic compounds has been well documented. Over 200 P450s have been identified, purified, and characterized, and they can be divided into many different gene families. These proteins have been found in essentially all aerobic organisms from prokaryotes to mammals. All P450s which have been studied can be divided into two groups on the basis of the electron transfer partner which is involved in the provision of the required electrons for the functioning of P450. The P450cam enzyme system has over the years proved to be an extremely powerful tool for structure–function studies. The data accumulated have been valuable for predicting how mammalian P450s might react during substrate binding, electron transfer, and oxygen activation. In addition, the three-dimensional structure of both classes of membrane-bound P450s has been predicted on the basis of sequence similarities to P450cam.