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. 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. Many proteins are modified at cysteine residues by fatty acids (usually palmitate). This modification imparts a local hydrophobic character to the protein and is crucial for diverse biological functions involving membrane-protein or protein-protein interactions, including vesicular transport, signal transduction, and maintenance of cellular architecture (reviewed in 1Smotrys J.E. Linder M.E. Palmitoylation of intracellular signaling proteins: regulation and function.Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (476) Google Scholar, 2Resh M.D. Membrane targeting of lipid modified signal transduction proteins.Subcell. Biochem. 2004; 37: 217-232Crossref PubMed Scopus (174) Google Scholar, 3Huang K. El-Husseini A. Modulation of neuronal protein trafficking and function by palmitoylation.Curr. Opin. Neurobiol. 2005; 15: 527-535Crossref PubMed Scopus (117) Google Scholar). Notable modified proteins include the transferrin receptor (4Jing S. Trowbridge I.S. Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site.EMBO J. 1987; 6: 327-331Crossref PubMed Scopus (144) Google Scholar), nitric oxide synthase (5Robinson L.J. Busconi L. Michel T. Agonist-modulated palmitoylation of endothelial nitric oxide synthase.J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), acetylcholinesterase (6Randall W.R. Cellular expression of a cloned, hydrophilic, murine acetylcholinesterase: evidence of palmitoylated membrane-bound forms.J. Biol. Chem. 1994; 269: 12367-12374Abstract Full Text PDF PubMed Google Scholar), G-protein α subunits, many G-protein-coupled receptors such as the adrenergic, serotonergic, and dopaminergic receptors, and cell-signaling molecules such as Src family protein tyrosine kinases and H- and N-p21Ras proteins (reviewed in 7Dunphy J.T. Linder M.E. Signalling functions of protein palmitoylation.Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (315) Google Scholar, 8Milligan G. Parenti M. Magee A.I. The dynamic role of palmitoylation in signal transduction.Trends Biochem. Sci. 1995; 20: 181-186Abstract Full Text PDF PubMed Scopus (284) Google Scholar). Neuronal proteins such as neuronal growth-associated protein-43, synaptosomal protein SNAP-25, and postsynaptic density-95 protein are also known to be palmitoylated. Cycles of palmitoylation and depalmitoylation have been described for some of these proteins, but the relevant enzyme(s) that mediates palmitate turnover in any of these processes has not been isolated or fully characterized (reviewed in 3Huang K. El-Husseini A. Modulation of neuronal protein trafficking and function by palmitoylation.Curr. Opin. Neurobiol. 2005; 15: 527-535Crossref PubMed Scopus (117) Google Scholar). A leading candidate deacylating enzyme that would operate in the cytosol is acyl-protein thioesterase (9Duncan J.A. Gilman A.G. A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS).J. Biol. Chem. 1998; 273: 15830-15837Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Overexpression of acyl-protein thioesterase accelerates the turnover of palmitate bound to G protein α subunits (10Duncan J.A. Gilman A.G. Characterization of Saccharomyces cerevisiae acyl-protein thioesterase 1, the enzyme responsible for G protein alpha subunit deacylation in vivo.J. Biol. Chem. 2002; 277: 31740-31752Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Palmitoylated proteins must also eventually undergo depalmitoylation in the course of their degradation and disposal in the lysosome. To date, only one enzyme has been described that plays a key role in the lysosomal metabolism of S-acylated proteins, a palmitoyl-protein thioesterase (PPT1). PPT1 (EC 3.1.2.22; also known as palmitoyl-protein hydrolase) is a lysosomal enzyme that removes fatty acids from their covalent thioester linkage to cysteine residues in S-acylated proteins. Mutations in this enzyme cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis (INCL) (11Vesa J. Hellsten E. Verkruyse L.A. Camp L.A. Rapola J. Santavuori P. Hofmann S.L. Peltonen L. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis.Nature. 1995; 376: 584-587Crossref PubMed Scopus (624) Google Scholar). This disorder is characterized by normal early development to the age of 18 months, followed by cortical and retinal atrophy. Clinical manifestations include motor and cognitive decline, seizures, and blindness, with death in the first decade of life. As in other forms of neuronal ceroid lipofuscinosis (NCL), autofluorescent storage material is readily demonstrated by light microscopy. The infantile form is distinguished from other forms of NCL by the characteristic electron microscopic appearance of the storage bodies, which are homogeneously granular and osmiophilic in appearance [granular osmiophilic deposits (GRODs)] (12Goebel H.H. Wisniewski K.E. Current state of clinical and morphological features in human NCL.Brain Pathol. 2004; 14: 61-69Crossref PubMed Scopus (153) Google Scholar). PPT1 was first purified from bovine brain (30,000-fold enrichment) on the basis of its ability to cleave palmitate from a model 3H-palmitoylated substrate, H-Ras (13Camp L.A. Hofmann S.L. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras.J. Biol. Chem. 1993; 268: 22566-22574Abstract Full Text PDF PubMed Google Scholar, 14Camp L.A. Verkruyse L.A. Afendis S.J. Slaughter C.A. Hofmann S.L. Molecular cloning and expression of palmitoyl-protein thioesterase.J. Biol. Chem. 1994; 269: 23212-23219Abstract Full Text PDF PubMed Google Scholar). A number of other S-acylated proteins and peptides are substrates, including palmitoylated Gα proteins (14Camp L.A. Verkruyse L.A. Afendis S.J. Slaughter C.A. Hofmann S.L. Molecular cloning and expression of palmitoyl-protein thioesterase.J. Biol. Chem. 1994; 269: 23212-23219Abstract Full Text PDF PubMed Google Scholar). Fatty acyl-CoAs are also substrates, allowing the chain length specificity of the enzyme to be determined; the fatty acid chain length optimum is between 14 and 18 carbons (14Camp L.A. Verkruyse L.A. Afendis S.J. Slaughter C.A. Hofmann S.L. Molecular cloning and expression of palmitoyl-protein thioesterase.J. Biol. Chem. 1994; 269: 23212-23219Abstract Full Text PDF PubMed Google Scholar). No detectable hydrolysis is observed for chain lengths <8 or >22 carbons. The turnover number of the enzyme is low (0.44 per second using palmitoyl-CoA as substrate) but similar to that of other lipases. The Km for palmitoyl-CoA and for palmitoylcysteine is similar (50 μM) (15Calero G. Gupta P. Nonato M.C. Tandel S. Biehl E.R. Hofmann S.L. Clardy J. 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. 2003; 278: 37957-37964Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The pH optimum varies with the substrate; it is near neutrality with H-Ras but in the range of 4 to 5 with the artificial substrate 4-methylumbelliferyl-S-palmitoyl-β-glucoside (16Voznyi Y.V. Keulemans J.L. Mancini G.M. Catsman-Berrevoets C.E. Young E. Winchester B. Kleijer W.J. van Diggelen O.P. A new simple enzyme assay for pre- and postnatal diagnosis of infantile neuronal ceroid lipofuscinosis (INCL) and its variants.J. Med. Genet. 1999; 36: 471-474Crossref PubMed Google Scholar). The 37 kDa, 306 amino acid enzyme contains motifs characteristic of other thioesterases (a G-X-S-X-G in the N-terminal half of the protein and a G-D-H near the C terminus). The localization of the gene encoding PPT1 to human chromosome 1p32 led to the recognition of its involvement in INCL (11Vesa J. Hellsten E. Verkruyse L.A. Camp L.A. Rapola J. Santavuori P. Hofmann S.L. Peltonen L. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis.Nature. 1995; 376: 584-587Crossref PubMed Scopus (624) Google Scholar). The X-ray crystallographic structure of PPT1 (determined with and without bound palmitate) has provided insights into the molecular basis for the phenotypes associated with known PPT1 mutations (17Bellizzi 3rd, J.J. Widom J. Kemp C. Lu J.Y. Das A.K. Hofmann S.L. Clardy J. The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 4573-4578Crossref PubMed Scopus (128) Google Scholar). The enzyme is a globular protein with a classical α/β hydrolase fold typical of lipases. The palmitate rests in a hydrophobic groove down the center of the enzyme; the peptide binding pocket is also easily discernible (Fig. 1). The classical hydrolytic catalytic triad consists of serine-115, aspartate-233, and histidine-289. Not surprisingly, mutations that affect residues near the active site and in the hydrophobic core of the enzyme are associated with a severe phenotype, whereas mutations in the binding pocket or at the periphery of the enzyme allow for residual activity and are associated with late-onset disease (Fig. 2). Serine is the active site nucleophile, yet like many lipases, the enzyme is resistant to inhibition by the alkylating agent PMSF. This was determined to be attributable to steric hindrance by the phenyl ring at the active site cleft (18Das A.K. Bellizzi 3rd, J.J. Tandel S. Biehl E. Clardy J. Hofmann S.L. Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride.J. Biol. Chem. 2000; 275: 23847-23851Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The enzyme is readily inactivated by a serine-reactive alkylating agent (hexadecylsulfonyl fluoride) that mimics the lipid substrate. Many lipases (including PPT2, a lysosomal palmitoyl-CoA hydrolase homologous to PPT1) possess a movable lid domain that regulates interfacial activation of the enzyme. However, PPT1 does not have such a lid; the crystallographic evidence suggests that the palmitate binding surface cleft is stabilized in the permanently open conformation (17Bellizzi 3rd, J.J. Widom J. Kemp C. Lu J.Y. Das A.K. Hofmann S.L. Clardy J. The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 4573-4578Crossref PubMed Scopus (128) Google Scholar).Fig. 2Mutations in PPT1 causing neuronal ceroid lipofuscinosis (NCL). Sites of clinical NCL mutations in PPT1 are mapped onto the peptide backbone. Infantile-onset mutations are displayed in red, a mutation causing late-infantile NCL symptoms is shown in blue, and juvenile-onset NCL mutations are shown in green. The most severe mutations (green) are found near the active site or within the hydrophobic core of the enzyme and would affect proper folding. Reproduced from (17Bellizzi 3rd, J.J. Widom J. Kemp C. Lu J.Y. Das A.K. Hofmann S.L. Clardy J. The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 4573-4578Crossref PubMed Scopus (128) Google Scholar). Copyright 2000 National Academy of Sciences, U.S.A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Most soluble lysosomal enzymes are targeted to lysosomes through a phosphate modification on mannose residues of asparagine-linked oligosaccharides via binding to the mannose 6-phosphate receptor. The binding may occur either within the secretory pathway or from the cell surface, as in the case of exogenously added lysosomal enzymes. The lysosomal targeting of PPT1 occurs through this classical mannose 6-phosphate receptor pathway, at least in peripheral tissues (19Verkruyse L.A. Hofmann S.L. Lysosomal targeting of palmitoyl-protein thioesterase.J. Biol. Chem. 1996; 271: 15831-15836Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 20Hellsten E. Vesa J. Olkkonen V.M. Jalanko A. Peltonen L. Human palmitoyl protein thioesterase: evidence for lysosomal targeting of the enzyme and disturbed cellular routing in infantile neuronal ceroid lipofuscinosis.EMBO J. 1996; 15: 5240-5245Crossref PubMed Scopus (149) Google Scholar, J. P. brain contains of including lysosomal enzymes and palmitoyl-protein enzyme in infantile neuronal Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). PPT1 has asparagine-linked at least two of which have been shown to the mannose 6-phosphate modification and M. P. Identification of of mannose on lysosomal Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The role of PPT1 in the lysosomal degradation of acylated proteins has been demonstrated through metabolic J.Y. Verkruyse L.A. Hofmann S.L. from acylated proteins in infantile neuronal ceroid of the in by palmitoyl-protein Natl. Acad. Sci. USA. 1996; PubMed Scopus Google Scholar, J.Y. Verkruyse L.A. Hofmann S.L. The of on normal and INCL evidence for the lysosomal of palmitoyl-protein thioesterase Biophys. Acta. 2002; PubMed Scopus Google Scholar). of from to the of a number of fatty acid and their is by of the with that the material fatty acylated is from proteins. also their PPT1 in the lysosomal metabolism of proteins. PPT1 or many features of the human disease P. A. Wisniewski K.E. J.A. Hofmann S.L. of PPT1 or causes neuronal ceroid lipofuscinosis in Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, A. Vesa J. T. C. T. Rapola J. M. with mutation the INCL and of 2005; PubMed Scopus Google Scholar). autofluorescent storage material in the brain and other tissues within and the storage material has the appearance characteristic of INCL The also and and by of using cortical neuronal from PPT1 evidence of a in number per with with a in as as in lysosomal whereas a number of and active properties of the are normal T. Gupta P. Hofmann S.L. in from neuronal ceroid 2005; 20: PubMed Scopus Google Scholar). The in number per would to early of and may key features of the clinical such as and The leading from enzyme deficiency to eventual neuronal death are only to be Neuronal cell death cortical is in the brain is a of normal and are lipidated peptides be found within the storage only a of its storage is not a of the and one only that the PPT1 are to that cell death substrate This in to lysosomal storage in which of of the enzyme is readily protein of the storage are A and which are not lipidated proteins but for other lysosomal enzymes J. M. M. of A and in infantile neuronal 1993; PubMed Scopus Google Scholar). is not for PPT1 to in INCL in other lysosomal storage to a in may L. K. M. J. Jalanko A. protein thioesterase 1 (PPT1) deficiency causes to PubMed Scopus Google Scholar). to neuronal death include in pH regulation T. Gupta P. Hofmann S.L. in from neuronal ceroid 2005; 20: PubMed Scopus Google Scholar, J. lysosomal pH in neuronal ceroid J. Biochem. 268: PubMed Scopus Google Scholar), of A. Vesa J. T. C. T. Rapola J. M. with mutation the INCL and of 2005; PubMed Scopus Google Scholar), and of the protein with activation of pathways S.J. P. A. deficiency mediates the activation of the protein and neuronal in Genet. 15: PubMed Scopus Google Scholar). of the of two and and activation of the and have been demonstrated in the of PPT1 S.J. P. A. deficiency mediates the activation of the protein and neuronal in Genet. 15: PubMed Scopus Google Scholar). of of and have also been S.J. deficiency to the activation of and to in Genet. 15: PubMed Scopus (51) Google Scholar). A of the metabolism of has been through a of from the in L. W.R. and of a prenylcysteine lyase from bovine Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, W.R. L. and cellular localization of a human prenylcysteine Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, W.R. J.A. Lysosomal prenylcysteine lyase is a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. and of human prenylcysteine Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, A. J.A. W.R. C.A. M. Young lyase deficiency in in the of and in brain and Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of or with bovine brain was found to and the cysteine be from substrate by The activity this was found in soluble and and was purified from bovine brain in a of by L. W.R. and of a prenylcysteine lyase from bovine Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The purified prenylcysteine lyase (EC also known as prenylcysteine as a on The Km for and is and is a for of the enzyme a amino for substrate prenyl peptides and are not it that prenylated proteins must be completely to prenylcysteine PCL A human encoding the enzyme was the amino acid of the purified bovine enzyme W.R. L. and cellular localization of a human prenylcysteine Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). which was with expression in The open is encoding a N-terminal amino acid signal peptide was as as asparagine-linked at residues and are by the and the enzyme in is Lysosomal localization was shown by of bovine and by of PCL with lysosomal in A of PCL to be a novel W.R. J.A. Lysosomal prenylcysteine lyase is a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. and of human prenylcysteine Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The was shown to be the aldehyde or Molecular was as a A bound is in the which is in not or A of is during the course of the The that for of the is shown in The of a without at to be and J.A. and of human prenylcysteine Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and to the of The as understood, is a that through a mechanism. The is by of the at of to (Fig. This a and a of a on the in the of a which forms the with to is one of the the of the The of the is to molecular to form the for the catalytic with targeted of the PCL gene demonstrated A. J.A. W.R. C.A. M. Young lyase deficiency in in the of and in brain and Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), PCL activity in was and to in in and in brain the of and to of these not from of was not and many human lysosomal storage the in not the of a human the in between and the of storage A that is to PCL amino acids is on human chromosome No concerning this protein is is that residual PCL activity from this or this have been in the human for PCL at is on the synthesis of proteins, including the of proteins in synthesis and proteins in the that processes the protein and of the to protein (reviewed in B. S. M. G. and for lipid and to 2003; PubMed Scopus Google Scholar). the metabolism of proteins has been may be from their through the of or a C (reviewed in G. protein in the regulation of Biochem. 2004; 37: PubMed Scopus Google Scholar). activity is in S. J. A in human 1987; PubMed Scopus Google Scholar, A for the of proteins is in Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, M.C. is that in is associated with J. Biochem. PubMed Scopus Google Scholar), and and is by J. T. A for of soluble a protein on human in regulation of and from and in of PubMed Scopus Google Scholar). The activity has also been described as in lysosomal of bovine T. E. P. of in J. 1996; PubMed Scopus Google Scholar). is into from and to a that is by the agent T. P. and intracellular of in Biophys. Acta. PubMed Scopus Google Scholar), which the of lysosomal of a function also by has been shown to be to and that lysosomal pH of on J. 2004; PubMed Scopus Google Scholar). However, was to the and lipid the lysosomal was not is to the of the intracellular metabolism of and proteins and a lysosomal is is to be a of proteins is a that has been The only one C is a cellular substrate of protein C in neuronal cell and regulation of the and lipid second signaling A. G. J. 2002; PubMed Scopus Google Scholar). of the by a soluble of brain has been S. A. of the substrate of protein C by the cytoplasmic of brain Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, G. S. T. C. The of C substrate and protein is in the Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). of the but form of was by and amino acid This was of the N-terminal also occur via between and least in T. G. N-terminal of the C substrate by cellular 2000; PubMed Scopus Google Scholar). by with brain be in The was to be to a within the substrate, as a for the and is by the of with is the activity in the cytosol or in a soluble lysosomal is that the is a function of in as of peptides by is a and be in the of A and in the regulation of bovine Biochem. 1996; PubMed Scopus Google Scholar). the of a lysosomal activity that mediates the of proteins is only at A enzyme has been described S. M. K. S. of peptide by and with enzyme Biophys. 1995; PubMed Scopus Google Scholar). pathway for the metabolism of from N-myristoylated proteins would be to the through the of monooxygenase Fatty acid a new role for enzyme and Biochem. Biophys. 1996; PubMed Scopus Google Scholar). The would be a substrate for the of fatty acid a membrane-bound enzyme and function of fatty acid Rev. Biochem. 2005; PubMed Scopus Google Scholar). is in such as and as are most lysosomal enzymes. is in lysosomes or in metabolism is open The lysosomal degradation of proteins two key PPT1 and enzymes are crucial for the metabolism of S-fatty acylated proteins and prenylated proteins, in PPT1 is a globular thioesterase that it the simple hydrolysis of palmitoylcysteine and palmitoylated and it is targeted to lysosomes through the classical mannose 6-phosphate receptor of PPT1 to a severe neurodegenerative disorder (INCL) in and PCL is a monooxygenase that metabolizes prenylcysteine but not prenylated and it is lysosomal PCL deficiency in known Much to be concerning the of that from PPT1 deficiency to neuronal cell are other tissues are and what or in PCL details of the eventual metabolic of and proteins are and may for future research.