Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice.

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.

Overexpression of low density lipoprotein (LDL) receptor eliminates LDL from plasma in transgenic mice.

A complementary DNA encoding the human low density lipoprotein (LDL) receptor under control of the mouse metallothionein-I promoter was injected into fertilized mouse eggs, and a strain of mice expressing high levels of LDL receptors was established. After administration of cadmium, these mice cleared intravenously injected 125I-labeled LDL from blood eight to ten times more rapidly than did normal mice. The plasma concentrations of apoproteins B-100 and E, the two ligands for the LDL receptor, declined by more than 90 percent after cadmium treatment, but the concentration of another apoprotein, A-I, was unaffected. Therefore, overexpression of an endocytotic receptor can dramatically lower the ambient concentration of its ligand in vivo.

Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice.

Lp(a) lipoprotein purified from human plasma bound with high affinity to isolated bovine LDL receptors on nitrocellulose blots and in a solid-phase assay. Lp(a) also competed with 125I-LDL for binding to human LDL receptors in intact fibroblasts. Binding led to cellular uptake of Lp(a) with subsequent stimulation of cholesterol esterification. After intravenous injection, human Lp(a) was cleared slowly from the plasma of normal mice. The clearance was markedly accelerated in transgenic mice that expressed large amounts of LDL receptors. We conclude that the covalent attachment of apo(a) to apo B-100 in Lp(a) does not interfere markedly with the ability of apo B-100 to bind to the LDL receptor and that this receptor has the potential to play a major role in clearance of Lp(a) from the circulation of intact humans.

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.

Mutations in the SHR5 gene of Saccharomyces cerevisiae suppress Ras function and block membrane attachment and palmitoylation of Ras proteins.

We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins.

Palmitoyl-protein thioesterase deficiency in fibroblasts of individuals with infantile neuronal ceroid lipofuscinosis and I-cell disease.

Mutations in the gene encoding a recently described lysosomal enzyme, palmitoyl-protein thioesterase (PPT), have recently been shown to result in the neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (INCL). Reduced palmitoyl-protein thioesterase enzyme has been demonstrated previously in INCL brain and immortalized lymphoblasts. In the current paper, we demonstrate that: (1) PPT can be detected by immunoblotting and enzyme activity assays in normal human skin fibroblasts; (2) INCL fibroblasts are deficient in PPT activity; (3) I-cell disease fibroblasts show markedly reduced intracellular levels of PPT but markedly increased levels of PPT in cell culture medium. These data establish that PPT is transported to lysosomes via the lysosomal enzyme:lysosomal enzyme receptor phosphomannosyl recognition system under normal physiological conditions and provide the basis for a useful clinical assay for INCL.

Identification of three novel mutations of the palmitoyl-protein thioesterase-1 (PPT1) gene in children with neuronal ceroid-lipofuscinosis.

Eight unrelated children with progressive neurological deterioration and granular osmiophilic deposits (GROD) due to an underlying palmitoyl-protein thioesterase deficiency were analyzed for mutations in the PPT1 gene. Three novel mutations (G118D, Q291X and F84del) were identified. The novel Q291X mutation was observed in an African-American child. The G118D and Q291X mutations occurred in infantile-onset subjects. These two mutations would be predicted to have severe effects on enzyme activity. The novel F84del mutation involves an invariant phenylalanine residue. A missense mutation, Q177E, occurred in three subjects from two families with late-infantile NCL, confirming an association of the Q177E mutation with a late-infantile phenotype. Other previously described mutations were R151X (5/16 alleles), T75P (3/16 alleles), R164X (1/16 alleles), and V181M (1/16 alleles). The current study expands the spectrum of mutations in PPT1 deficiency and further confirms the broad range of age of onset of symptoms resulting from an enzyme deficiency previously associated only with infantile NCL.

Infantile neuronal ceroid lipofuscinosis: no longer just a 'Finnish' disease.

The neuronal ceroid lipofuscinoses (NCLs) are a group of enigmatic neurodegenerative disorders of children that have in common the storage of autofluorescent lipofuscin, or aging pigment, in the brain. With the identification of the three major genes involved in the disorder, the NCLs are now appreciated to represent true lysosomal storage disorders. The most severe (infantile) form of NCL is caused by mutations in a lysosomal thioesterase that removes fatty acids from modified cysteine residues in proteins. Although the disorder was first described in Finland (and the identification of the underlying gene (CLN1) made in this population) defects in CLN1 and the underlying deficiency have been widely reported outside of Scandinavia. In this report, we summarize the relationship of genotype to phenotype in the disorder and evaluate known mutations in light of the recently solved crystal structure of defective enzyme, palmitoyl-protein thioesterase (PPT). We also discuss progress in identifying the fatty acyl cysteine thioesters that accumulate in PPT deficiency and in working toward animal models of NCL. Recent progress in these areas holds promise for the eventual treatment of the disorder.

Palmitoyl-protein thioesterase deficiency in a novel granular variant of LINCL.

Typically, late infantile neuronal ceroid-lipofuscinosis (LINCL) patients present between the ages of 2 and 4 years with progressive dementia, blindness, seizures, and motor dysfunction. Curvilinear profiles are seen on electron microscopic examination of tissues derived from those patients. Data were collected on 122 LINCL cases, representing 81 independent families, diagnosed on the basis of age of onset, clinical symptomatology, and pathologic findings. Careful analysis of our data has revealed that 20% of these cases (24 of 122) show either an atypical clinical course or atypical pathologic findings and may represent variants of LINCL. Recent progress in the biochemistry and molecular genetics of NCL has led us to reevaluate these atypical cases. Five atypical LINCL cases (representing three independent families) manifested granular inclusions when examined by electron microscopy, a finding normally associated with the infantile form of NCL. In addition, these five cases did not show elevated subunit c levels in urine (typically seen in LINCL). In these five cases, palmitoyl-protein thioesterase activity was found to be deficient (less than 10% normal activity), suggesting that these cases represent INCL, presenting at a later age of onset. These findings suggest that palmitoyl-protein thioesterase deficiency is not restricted to infantile onset cases, and they raise the possibility that milder forms of INCL may result from less deleterious mutations.

Retinoids--"differentiation agents" for cancer treatment and prevention.

The ability of vitamin A and its derivatives to induce differentiation in certain target tissues has been appreciated for nearly a century. Recently, oral all-trans retinoic acid (ATRA), a vitamin A metabolite, has been shown to induce terminal differentiation of leukemic cells in patients with acute promyelocytic leukemia (APL). Complete remissions are obtained and normal hematopoiesis is established in an outpatient setting with minimal side effects in the majority of cases. Although remissions are not durable, disseminated intravascular coagulation, a frequent complication of remission induction in APL, is avoided by oral ATRA prior to definitive chemotherapy. The molecular basis for the efficacy of ATRA in APL appears to be the involvement of the retinoic acid receptor alpha locus in the t(15;17) translocation breakpoint characteristic of APL.

Southwestern Internal Medicine Conference: Shiga-like toxins in hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura.

The majority of cases of hemolytic-uremic syndrome and a smaller proportion of cases of thrombotic thrombocytopenic purpura have recently been shown to result from a toxin produced by enteric bacteria, referred to as verotoxin, or Shiga-like toxin. The predominant toxin-producing bacterial strain in North America is E. coli O157:H7, which causes hemorrhagic colitis in humans after ingestion of contaminated meat. The toxin is believed to gain entry to the circulation from the bowel wall; it then binds to specific glycolipid receptors abundant on renal vascular endothelial cells. The toxin inactivates ribosomes inside the cells, thereby killing them and producing the clinical manifestations of hemolytic-uremic syndrome. Recognition of the etiology of hemolytic-uremic syndrome may lead to better prospects for prevention and treatment.

Inefficient cleavage of palmitoyl-protein thioesterase (PPT) substrates by aminothiols: implications for treatment of infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL, also known as infantile Batten disease) is a devastating neurodegenerative disorder caused by deficiency in the lysosomal enzyme palmitoyl-protein thioesterase (PPT, or CLN1), which functions to remove long-chain fatty acids from cysteine residues in proteins. A previous study suggested that the drug cysteamine, a simple aminothiol used in the treatment of cystinosis, may have utility in the treatment of INCL. In the current study, we compared the catalytic rate constants for the conversion of palmitoyl-CoA (a PPT substrate) and cystine (which accumulates in cystinosis) by cysteamine. We found that while cysteamine can react with palmitoyl-CoA, the rate constant is 10(3)-fold less than the reaction with cystine. Structure-activity studies suggested that it is the thiolate ion that is reactive in the cleavage reaction and that the amino group probably facilitates lysosomal entry. A modest effect of cysteamine (and two related aminothiols, WR 1065 and dimethylaminoethanethiol, DMAET) on PPT substrate accumulation in INCL lymphoblasts was observed. However, at optimum concentration a paradoxical increase in saposin immunoreactivity was seen, indicating possible lysosomal dysfunction. Improvements are needed in the design of small molecules for the treatment of INCL disease.

Depalmitoylation of CAAX motif proteins. Protein structural determinants of palmitate turnover rate.

In the present study, we examined the effect of amino acid substitutions on the rate of turnover of palmitate bound to a model "CAAX" motif protein H-Ras. These experiments were designed to shed light on the specificity of the process that removes palmitate from prenylated proteins. H-Ras, protein A-Ras fusion constructs, and constructs with amino acid substitutions in the H-Ras hypervariable region were transfected into COS cells, and the turnover rate of palmitate bound to each expressed protein was measured. We found no evidence for strict sequence specificity for palmitate removal, but found a strong inverse correlation between palmitate turnover rate and the degree of membrane association for any given construct, with slower turnover rates associated with stronger membrane binding. These data support a model in which the palmitate turnover rate is determined by access to a depalmitoylating enzyme and argue against a more complex model in which specific recognition of palmitoylated proteins is required.

The neuronal ceroid-lipofuscinoses (Batten disease): a new class of lysosomal storage diseases.

The neuronal ceroid-lipofuscinoses (Batten disease) are a group of severe neurodegenerative disorders characterized clinically by visual loss, seizures and psychomotor degeneration, and pathologically by loss of neurons and lysosomal accumulation of autofluorescent storage material resembling ageing pigment. To date, eight genetic loci have been identified (CLN1-8). Four CLN genes have been isolated (CLN1, CLN2, CLN3 and CLN5) and their gene products have been characterized. CLN1 is a lysosomal palmitoyl-protein thioesterase (PPT) and CLN2 is a lysosomal pepstatin-insensitive peptidase. CLN3 and CLN5 are proteins with multiple membrane-spanning regions and have no homologies to other proteins that would suggest their function. The CLN3 protein is associated with lysosomal membranes and the intracellular location of the CLN5 protein is unknown. Therefore, there is ample evidence that the neuronal ceroid-lipofuscinoses represent a new class of lysosomal storage disorders.

Lysosomal targeting of palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a newly described long chain fatty-acid hydrolase that removes fatty acyl groups from modified cysteines in proteins. We have recently identified palmitoyl-protein thioesterase as the defective enzyme in the recessive hereditary neurological degenerative disorder infantile neuronal ceroid lipofuscinosis (Vesa, J., Hellsten, E., Verkruyse, L. A., Camp, L. A. , Rapola, J., Santavuori, P., Hofmann, S. L., and Peltonen, L. (1995) Nature 376, 584-587). A defect in a lysosomal enzyme had been postulated for the disease, but until recently, the relevant defective lysosomal enzyme had not been identified. In this paper, we present evidence for the lysosomal localization of palmitoyl-protein thioesterase. We show that COS cells take up exogenously supplied palmitoyl-protein thioesterase intracellularly and that the cellular uptake is blocked by mannose 6-phosphate, a hallmark of lysosomal enzyme trafficking. The enzyme contains endoglycosidase H-sensitive oligosaccharides that contain phosphate groups. Furthermore, palmitoyl-protein thioesterase cosediments with lysosomal enzyme markers by Percoll density gradient centrifugation. Interestingly, the pH optimum for the enzyme is in the neutral range, a property shared by two other lysosomal enzymes that remove post-translational protein modifications. These findings suggest that palmitoyl-protein thioesterase is a lysosomal enzyme and that infantile neuronal ceroid lipofuscinosis is properly classified as a lysosomal storage disorder.

Molecular cloning and expression of palmitoyl-protein thioesterase 2 (PPT2), a homolog of lysosomal palmitoyl-protein thioesterase with a distinct substrate specificity.

Palmitoyl-protein thioesterase is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. Mutations in this enzyme were recently shown to underlie the hereditary neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis, and lipid thioesters derived from acylated proteins were found to accumulate in lymphoblasts from individuals with the disorder. In the current study, we describe the cloning and expression of a second lysosomal thioesterase, palmitoyl-protein thioesterase 2 (PPT2), that shares an 18% identity with palmitoyl-protein thioesterase. Transient expression of a PPT2 cDNA led to the production of a glycosylated lysosomal protein with palmitoyl-CoA hydrolase activity comparable with palmitoyl-protein thioesterase. However, PPT2 did not remove palmitate groups from palmitoylated proteins that are substrates for palmitoyl-protein thioesterase. In cross-correction experiments, PPT2 did not abolish the accumulation of protein-derived lipid thioesters in palmitoyl-protein thioesterase-deficient cell lines. These results indicate that PPT2 is a lysosomal thioesterase that possesses a substrate specificity that is distinct from that of palmitoyl-protein thioesterase.

Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras.

H-Ras, the protein product of the cellular homologue of the Harvey ras oncogene, undergoes a complex series of post-translational modifications that include C-terminal isoprenylation, proteolysis, methylation, and palmitoylation. Palmitoylation has been shown to enhance the transformation efficiency of H-Ras about 10-fold in vivo. A recent study (Magee, A. I., Gutierrez, L., McKay, I. A., Marshall, C. J., and Hall, A. (1987) EMBO J. 6, 3353-3357) has provided strong evidence that the palmitate undergoes a dynamic acylation-deacylation cycle, but details concerning the enzymology of this process and its regulation are lacking. To begin to dissect this event, we have developed an assay for the enzymatic removal of palmitate from [3H]palmitate-labeled H-Ras. This substrate was produced in a baculovirus expression system and was used to purify to homogeneity a novel 37-kDa enzyme from bovine brain cytosol that removes the radiolabeled palmitate. The purified enzyme is sensitive to diethyl pyrocarbonate and insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. Interestingly, the thioesterase recognizes H-Ras as a substrate only when H-Ras is in its native conformation (bound to Mg2+ and guanine nucleotide). The palmitoylated alpha subunits of the heterotrimeric G proteins are also substrates for the enzyme.