Glycosylation, transport, and complex formation of palmitoyl protein thioesterase 1 (PPT1) – distinct characteristics in neurons

Lyly, Annina; Schantz, Carina von; Salonen, Tarja; Kopra, Outi; Saarela, Jani; Jauhiainen, Matti; Kyttälä, Aija; Jalanko, Anu

doi:10.1186/1471-2121-8-22

Cited by 40 publications

(64 citation statements)

References 51 publications

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“…Western blotting with an anti-PPT1 antibody confirmed a similar migration pattern for hPPT1 protein in transfected cell lysates (Figure 3D, bottom panel). These data indicate that both ABC34 ( 13 ) and ABC45 react with PPT1 and are consistent with previous studies demonstrating that this enzyme is glycosylated (Camp et al, 1994; Lyly et al, 2007). In contrast, FP-Rh failed to detect hPPT1 in transfected cell lysates (Figure 3D).…”

Section: Resultssupporting

confidence: 92%

“…(D) Verification that recombinant human PPT1 expressed by transient transfection in HEK293T cells reacts with ABC45 (1 µM), but not FP-Rh (1 µM), and the ABC45 reactivity is blocked by ABC34 (5 µM, 4 h). PNGaseF treatment causes a shift in PPT1 signals by SDS-PAGE, consistent with the reported glycosylation state of this protein (Camp et al, 1994; Lyly et al, 2007). Upper, ABPP gel; lower, anti-PPT1 western blot.…”

Section: Figuresupporting

confidence: 87%

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Selective N-Hydroxyhydantoin Carbamate Inhibitors of Mammalian Serine Hydrolases

Cognetta

Niphakis

Lee

et al. 2015

Chemistry & Biology

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Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis and screening of a small library of NHH carbamates by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterease-1 (PPT1), mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis.

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Section: Resultssupporting

confidence: 92%

Section: Figuresupporting

confidence: 87%

Selective N-Hydroxyhydantoin Carbamate Inhibitors of Mammalian Serine Hydrolases

Cognetta

Niphakis

Lee

et al. 2015

Chemistry & Biology

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“…In addition to an MPR-independent route, Cathepsin D can also use the classical MPR pathway for its lysosomal trafficking [Canuel et al, 2008;Kornfeld and Mellman, 1989;von Figura and Hasilik, 1986]. Palmitoyl protein thioesterase 1 (PPT1/CLN1), the protein defective in the infantile NCL, has also been reported to show properties that differ from those involved in the classical MPRmediated sorting [Lyly et al, 2007]. Interestingly, we have recently shown that PPT1/CLN1 interacts with CLN5, and furthermore, can facilitate the trafficking of the mutated CLN5 from the ER to the lysosomes [Lyly et al, 2009].…”

Section: Discussionmentioning

confidence: 98%

The neuronal ceroid lipofuscinosis protein CLN5: new insights into cellular maturation, transport, and consequences of mutations

et al. 2010

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Neuronal ceroid lipofuscinoses (NCLs) represent a group of children's inherited neurodegenerative disorders caused by mutations in at least eight different genes. Mutations in the CLN5 gene result in the Finnish variant late infantile NCL characterized by gradual loss of vision, epileptic seizures, and mental deterioration. The CLN5 gene encodes a lysosomal glycoprotein of unidentified function. In this study, we have used both transient and stable expression systems for the characterization of CLN5, focusing on the localization, processing, and intracellular trafficking. We show that CLN5 is proteolytically cleaved, and that the mature polypeptide is transported to the lysosomes. Our data provide the first evidence that soluble CLN5 protein can also undergo mannose-6-phosphate receptor-independent trafficking to the lysosomes. We studied the localization and maturation of the CLN5 carrying the previously uncharacterized vLINCL disease causing mutations in HeLa cells. All analyzed disease mutations disturb the lysosomal trafficking of the mutated CLN5 proteins. The level of lysosomal targeting does not correlate, however, to disease onset, indicating that CLN5 may also function outside lysosomes. This study furthers our understanding of the basic properties of the CLN5 protein, necessary for the characterization of the consequences of disease mutations and for the planning of future therapies for vLINCL.

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“…The glycan moieties are endoglycosidase H sensitive, demonstrating that PPT1 possesses only N-type glycosylation. Proper glycosylation is required for the stability and activity of the enzyme 30 and glycosylation site mutants of PPT1 have greatly reduced enzyme activity 44 . Interestingly, despite localization of PPT1 in lysosomes, its enzymatic activity is optimal at neutral pH not pH 4-5 40 (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Identification of Palmitoyl Protein Thioesterase 1 in Human THP1 Monocytes and Macrophages and Characterization of Unique Biochemical Activities for This Enzyme

et al. 2013

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The profiles of serine hydrolases in human and mouse macrophages are similar yet different. For instance, human macrophages express high levels of carboxylesterase 1 (CES1), whereas mouse macrophages have minimal amounts of the orthologous murine CES1. On the other hand, both species' macrophages exhibit limited expression of the canonical 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, MAGL. Our previous study showed carboxylesterase 1 (CES1) was partly responsible for the hydrolysis of 2-AG (50%) and prostaglandin glyceryl esters (PG-Gs) (80-95%) in human THP1 monocytes/macrophages. However, MAGL and other endocannabinoid hydrolases, FAAH, ABHD6 and ABHD12, did not have a role because of either limited or no expression. Thus, another enzyme was hypothesized to be responsible for the remaining 2-AG hydrolysis activity following chemical inhibition and immunodepletion of CES1 (previous study) or CES1 gene knockdown (this study). Here we identified two candidate serine hydrolases in THP1 cell lysates by activity-based protein profiling (ABPP)–MudPIT and western blotting: cathepsin G and palmitoyl protein thioesterase 1 (PPT1). Both proteins exhibited similar electrophoretic properties to a serine hydrolase in THP1 cells detected by gel-based ABPP at 31-32 kDa; however, only PPT1 exhibited lipolytic activity and hydrolyzed 2-AG in vitro. Interestingly, PPT1 was highly expressed in THP1 cells but was significantly less reactive than cathepsin G toward the activity-based probe, fluorophosphonate-biotin. KIAA1363, another serine hydrolase, was also identified in THP1 cells but did not have significant lipolytic activity. On the basis of chemoproteomic profiling, immunodepletion studies and chemical inhibitor profiles, we estimated that PPT1 contributed 32-40% of 2-AG hydrolysis activity in the THP1 cell line. In addition, pure recombinant PPT1 catalyzed the hydrolysis of 2-AG, PGE2-G and PGF2α-G, although the catalytic efficiency of 2-AG hydrolysis by PPT1 was ∼10-fold lower than CES1's. PPT1 was also insensitive to several chemical inhibitors that potently inhibit CES1, such as organophosphate poisons and JZL184. This is the first report to document the expression of PPT1 in a human monocyte/macrophage cell line and to show PPT1 can hydrolyze the natural substrates 2-AG and PG-Gs. These findings suggest that PPT1 may participate in endocannabinoid metabolism within specific cellular contexts, and highlights the functional redundancy often exhibited by enzymes involved in lipid metabolism.

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Glycosylation, transport, and complex formation of palmitoyl protein thioesterase 1 (PPT1) – distinct characteristics in neurons

Cited by 40 publications

References 51 publications

Selective N-Hydroxyhydantoin Carbamate Inhibitors of Mammalian Serine Hydrolases

Selective N-Hydroxyhydantoin Carbamate Inhibitors of Mammalian Serine Hydrolases

The neuronal ceroid lipofuscinosis protein CLN5: new insights into cellular maturation, transport, and consequences of mutations

Identification of Palmitoyl Protein Thioesterase 1 in Human THP1 Monocytes and Macrophages and Characterization of Unique Biochemical Activities for This Enzyme

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