A Rapid Fluorogenic Palmitoyl-Protein Thioesterase Assay: Pre- and Postnatal Diagnosis of INCL

Diggelen, O. P. van; Keulemans, J. L. M.; Winchester, Bryan; Hofman, I. L.; Vanhanen, Sanna‐Leena; Santavuori, Pirkko; Voznyi, Ya. V.

doi:10.1006/mgme.1999.2809

Cited by 113 publications

(90 citation statements)

References 12 publications

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“…PPT1 prefers acyl chain lengths of 14 -18 carbons (6), and no appreciable hydrolysis is observed for chain lengths under eight carbons. Previous work has also shown that there are no major constraints on the nature of the attached "leaving group," because PPT1 readily hydrolyzes other long chain fatty acyl thioesters, such as palmitoyl-CoA (6) and S-palmitoyl thioglucoside (19). The current studies presented below utilized palmitoyl CoA as a substrate because of the availability of facile assays using [ Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Das¹,

Bellizzi²,

Tandel³

et al. 2000

Journal of Biological Chemistry

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Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K i of the inhibition was 125 M (in the presence of 1.5 mM Triton X-100), and the catalytic rate constant for sulfonylation (k 2 ) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 Å resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.Palmitoyl-protein thioesterase-1 (PPT1) 1 is a newly described lysosomal hydrolase that removes long chain fatty acids from lipid-modified cysteine residues in fatty acylated proteins (reviewed in Ref. 1). Deficiency of the enzyme leads to a lysosomal storage disease, infantile neuronal ceroid lipofuscinosis, which causes blindness, seizures, and cortical atrophy of the brain (2). Lysosomal inclusion bodies, termed granular osmiophilic deposits, accumulate in all tissues, and resemble lipofuscin deposits that occur during normal aging. In keeping with this important lipid-metabolizing role, PPT1 is one of the most abundant lysosomal enzymes in the brain (3). In contrast to most hydrolytic enzymes (such as proteases, lipases, esterases, and thioesterases), PPT1 is insensitive to the serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) (4). The insensitivity of PPT1 to these reagents was an important feature of PPT1 because it allowed for the identification of PPT activity in the presence of contaminating lipase and protease activities. We wondered whether there might be structural and/or mechanistic differences between PPT1 and these other lipolytic enzymes that would account for this observation. We have recently determined the three-dimensional crystal structure of PPT1 in the presence and absence of palmitoyl-CoA and shown that palmitate modifies serine 115 of the enzyme (5), confirming that a serine residue is the catalytic nucleophile.In the current study, we show that although PPT1 is insensitive to inactivation by the ser...

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Section: Resultsmentioning

confidence: 99%

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Das¹,

Bellizzi²,

Tandel³

et al. 2000

Journal of Biological Chemistry

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“…Palmitoyl-protein-thioesterase-and tripeptidylpeptidase-I enzyme activities were determined by using the f luorogenic substrates MU-6S-palm-␤Glc (4-methylumbelliferyl-6-thiopalmitoyl ␤-D-galactopyranoside) (Moscerdam, Rotterdam, The Netherlands) and AAF-AMC (Ala-Ala-Phe-7-amido-4-methylcoumarin) (Sigma), respectively, and the assays were performed twice in triplicate as described (20)(21)(22).…”

Section: Methodsmentioning

confidence: 99%

Neuronal loss and brain atrophy in mice lacking cathepsins B and L

Felbor

Kessler²,

Mothes³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

305

217

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Cathepsins B and L are widely expressed cysteine proteases implicated in both intracellular proteolysis and extracellular matrix remodeling. However, specific roles remain to be validated in vivo. Here we show that combined deficiency of cathepsins B and L in mice is lethal during the second to fourth week of life. Cathepsin B ؊/؊ ͞L ؊/؊ mice reveal a degree of brain atrophy not previously seen in mice. This is because of massive apoptosis of select neurons in the cerebral cortex and the cerebellar Purkinje and granule cell layers. Neurodegeneration is accompanied by pronounced reactive astrocytosis and is preceded by an accumulation of ultrastructurally and biochemically unique lysosomal bodies in large cortical neurons and by axonal enlargements. Our data demonstrate a pivotal role for cathepsins B and L in maintenance of the central nervous system.

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“…Hydrolysis of [ 3 H]palmitoyl-H-Ras was determined as described by Camp and Hofmann (6). Hydrolysis of the fluorescent substrate 4-methylumbelliferyl-6-S-palmitoyl-␤-D-glucoside was performed by the method of van Diggelen et al (12) and was quantified using a standard curve of increasing concentrations of 4-methylumbelliferone (Sigma).…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Calero

Gupta

Nonato

et al. 2003

Journal of Biological Chemistry

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Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform nonredundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 Å. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix ␣4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (␤3-␣A and ␤8-␣F) located immediately above the lipidbinding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.

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A Rapid Fluorogenic Palmitoyl-Protein Thioesterase Assay: Pre- and Postnatal Diagnosis of INCL

Cited by 113 publications

References 12 publications

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Neuronal loss and brain atrophy in mice lacking cathepsins B and L

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

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