The long-term goal of the original proposal, now in its second year, is to understand the mechanisms involved in protein deacylation by isolating and characterizing the enzyme(s) responsible for the turnover of palmitate covalently bound to a model palmitoylated protein, H-Ras. In the past two years, we have purified and molecularly cloned palmitoyl-protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and other proteins in vitro. From these experiments, we learned that PPT is secreted from cells and is unlikely to play a role in Ras plamitate turnover. However, we have recently demonstrated that mutations in the PPT gene cause the recessively-inherited neuronal degenerative disease, infantile neuronal ceroid lipofuscinosis (INCL). This disease is characterized by the accumulation of proteolipid material in storage bodies within cells, suggesting that PPT plays a crucial role in the catabolism of lipid-modified proteins. In the next funding period, we will continue to focus upon the molecular and cell biology of this novel enzyme. We propose to characterize the PPT gene, to explore protein structure-function relationships through the study of naturally-occurring and site-directed mutations, and to determine how the enzyme trafficks within the cell. We will also use cells derived from normal and INCL patients in metabolic labeling experiments to test the hypothesis that PPT plays a role in the catabolism of lipid-modified proteins. Finally, we will analyze the products of the enzymatic reaction of PPT and INCL cells or cell extracts to identify the most significant physiological substrates for PPT. We believe that this work may lead to new insights into cellular metabolism and the pathogenesis of neurodegenerative disease.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-NLS-3 (01))
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Small, Judy A
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University of Texas Sw Medical Center Dallas
Internal Medicine/Medicine
Schools of Medicine
United States
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Cho, Steve K; Hofmann, Sandra L (2004) pdf1, a palmitoyl protein thioesterase 1 Ortholog in Schizosaccharomyces pombe: a yeast model of infantile Batten disease. Eukaryot Cell 3:302-10
Calero, Guillermo; Gupta, Praveena; Nonato, M Cristina et al. (2003) The crystal structure of palmitoyl protein thioesterase-2 (PPT2) reveals the basis for divergent substrate specificities of the two lysosomal thioesterases, PPT1 and PPT2. J Biol Chem 278:37957-64
Lu, Jui-Yun; Verkruyse, Linda A; Hofmann, Sandra L (2002) The effects of lysosomotropic agents on normal and INCL cells provide further evidence for the lysosomal nature of palmitoyl-protein thioesterase function. Biochim Biophys Acta 1583:35-44
Hofmann, Sandra L; Atashband, Armita; Cho, Steve K et al. (2002) Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomal enzymes (CLN1 and CLN2). Curr Mol Med 2:423-37
Rush, Jeffrey S; Cho, Steve K; Jiang, Songmin et al. (2002) Identification and characterization of a cDNA encoding a dolichyl pyrophosphate phosphatase located in the endoplasmic reticulum of mammalian cells. J Biol Chem 277:45226-34
Gupta, P; Hofmann, S L (2002) Neuronal ceroid lipofuscinosis/Batten disease: the lysosomal proteinoses. Mol Psychiatry 7:434-6
Hofmann, S L; Das, A K; Lu, J Y et al. (2001) Positional candidate gene cloning of CLN1. Adv Genet 45:69-92
Das, A K; Bellizzi 3rd, J J; Tandel, S et al. (2000) Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride. J Biol Chem 275:23847-51
Bellizzi 3rd, J J; Widom, J; Kemp, C et al. (2000) The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis. Proc Natl Acad Sci U S A 97:4573-8
Bennett, M J; Hofmann, S L (1999) The neuronal ceroid-lipofuscinoses (Batten disease): a new class of lysosomal storage diseases. J Inherit Metab Dis 22:535-44

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