Multiple mitochondrial thioesterases have distinct tissue and substrate specificity and CoA regulation, suggesting unique functional roles.

Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate ß-oxidation overload and maintain CoA availability. Several Acots associate with mitochondria, but whether they all localize to the matrix, are redundant, or have different roles is unresolved. Here, we compared the suborganellar localization, activity, expression, and regulation among mitochondrial Acots (Acot2, -7, -9, and -13) in mitochondria from multiple mouse tissues and from a model of Acot2 depletion. Acot7, -9, and -13 localized to the matrix, joining Acot2 that was previously shown to localize there. Mitochondria from heart, skeletal muscle, brown adipose tissue, and kidney robustly expressed Acot2, -9, and -13; Acot9 levels were substantially higher in brown adipose tissue and kidney mitochondria, as was activity for C4:0-CoA, a unique Acot9 substrate. In all tissues, Acot2 accounted for about half of the thioesterase activity for C14:0-CoA and C16:0-CoA. In contrast, liver mitochondria from fed and fasted mice expressed little Acot activity, which was confined to long-chain CoAs and due mainly to Acot7 and Acot13 activities. Matrix Acots occupied different functional niches, based on substrate specificity (Acot9 versus Acot2 and -13) and strong CoA inhibition (Acot7, -9, and -13, but not Acot2). Interpreted in the context of ß-oxidation, CoA inhibition would prevent Acot-mediated suppression of ß-oxidation, while providing a release valve when CoA is limiting. In contrast, CoA-insensitive Acot2 could provide a constitutive siphon for long-chain fatty acyl-CoAs. These results reveal how the family of matrix Acots can mitigate ß-oxidation overload and prevent CoA limitation.

Regulation of fatty acid trafficking in liver by thioesterase superfamily member 1.

Thioesterase superfamily member 1 (Them1) is an acyl-CoA thioesterase that is highly expressed in brown adipose tissue, where it functions to suppress energy expenditure. Lower Them1 expression levels in the liver are upregulated in response to high-fat feeding. Them1-/- mice are resistant to diet-induced obesity, hepatic steatosis, and glucose intolerance, but the contribution of Them1 in liver is unclear. To examine its liver-specific functions, we created conditional transgenic mice, which, when bred to Them1-/- mice and activated, expressed Them1 exclusively in the liver. Mice with liver-specific Them1 expression exhibited no changes in energy expenditure. Rates of fatty acid oxidation were increased, whereas hepatic VLDL triglyceride secretion rates were decreased by hepatic Them1 expression. When fed a high-fat diet, Them1 expression in liver promoted excess steatosis in the setting of reduced rates of fatty acid oxidation and preserved glycerolipid synthesis. Liver-specific Them1 expression did not influence glucose tolerance or insulin sensitivity, but did promote hepatic gluconeogenesis in high-fat-fed animals. This was attributable to the generation of excess fatty acids, which activated PPARα and promoted expression of gluconeogenic genes. These findings reveal a regulatory role for Them1 in hepatocellular fatty acid trafficking.

Inflammatory stimuli induce acyl-CoA thioesterase 7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages.

Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2 production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥.

Functional characterization of thioesterase superfamily member 1/Acyl-CoA thioesterase 11: implications for metabolic regulation.

Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is highly expressed in brown adipose tissue and limits energy expenditure in mice. Them1 is a putative fatty acyl-CoA thioesterase that comprises tandem hot dog-fold thioesterase domains and a lipid-binding C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. To better define its role in metabolic regulation, this study examined the biochemical and enzymatic properties of Them1. Purified recombinant Them1 dimerized in solution to form an active fatty acyl-CoA thioesterase. Dimerization was induced by fatty acyl-CoAs, coenzyme A (CoASH), ATP, and ADP. Them1 hydrolyzed a range of fatty acyl-CoAs but exhibited a relative preference for long-chain molecular species. Thioesterase activity varied inversely with temperature, was stimulated by ATP, and was inhibited by ADP and CoASH. Whereas the thioesterase domains of Them1 alone were sufficient to yield active recombinant protein, the START domain was required for optimal enzyme activity. An analysis of subcellular fractions from mouse brown adipose tissue and liver revealed that Them1 contributes principally to the fatty acyl-CoA thioesterase activity of microsomes and nuclei. These findings suggest that under biological conditions, Them1 functions as a lipid-regulated fatty acyl-CoA thioesterase that could be targeted for the management of metabolic disorders.

Novel CLN1 mutation in two Italian sibs with late infantile neuronal ceroid lipofuscinosis.

We detected a novel CLN1 mutation (c.125-15t>g) in two Italian siblings. The clinical phenotype is that of a variant late-infantile neuronal ceroid lipofuscinosis and consisted of early-onset visual loss, psychomotor deterioration, and seizures. Ultrastructurally, granular osmiophilic deposits were found in skin biopsy of both patients. The novel mutation occurs in the acceptor sequences for splicing and leads to skipping of multiple exons. This predicts a protein lacking part or all of the active site of the enzyme and the palmitate-binding pocket. Consequently, biochemical activity of the palmitoyl protein thioesterase-1 enzyme was drastically reduced. The new mutation was not identified in a large set of ethnically matched control chromosomes. Our findings support the notion that CLN1 patients are not rare in Southern Europe and facilitate DNA-based mutation and carrier testing in this family.

Autosomal dominant adult neuronal ceroid lipofuscinosis: a novel form of NCL with granular osmiophilic deposits without palmitoyl protein thioesterase 1 deficiency.

We describe the neuropathological and biochemical autopsy findings in 3 patients with autosomal dominant adult neuronal ceroid lipofuscinosis (ANCL, Parry type; MIM 162350), from a family with 6 affected individuals in 3 generations. Throughout the brain of these patients, there was abundant intraneuronal lysosomal storage of autofluorescent lipopigment granules. Striking loss of neurons in the substantia nigra was found. In contrast, little neuronal cell loss occurred in other cerebral areas, despite massive neuronal inclusions. Visceral storage was present in gut, liver, cardiomyocytes, skeletal muscle, and in the skin eccrine glands. The storage material showed highly variable immunoreactivity with antiserum against subunit c of mitochondrial ATP synthase, but uniform strong immunoreactivity for saposin D (sphingolipid activating protein D). Protein electrophoresis of isolated storage material revealed a major protein band of about 14 kDa, recognized in Western blotting by saposin D antiserum (but not subunit c of mitochondrial ATPase (SCMAS) antiserum). Electron microscopy showed ample intraneuronal granular osmiophilic deposits (GRODs), as occurs in CLN1 and congenital ovine NCL. These forms of NCL are caused by the deficiencies of palmitoyl protein thioesterase 1 and cathepsin D, respectively. However, activities of these enzymes were within normal range in our patients. Thus we propose that a gene distinct from the cathepsin D and CLN1-CLN8 genes is responsible for this autosomal dominant form of ANCL.

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood.

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.

Molecular prenatal diagnosis in families with fetal mitochondrial trifunctional protein mutations.

OBJECTIVES: To evaluate the feasibility of molecular prenatal diagnosis in families with mitochondrial trifunctional protein (TFP) mutations and prospectively study the effects of fetal genotype on pregnancy outcome. TFP catalyzes the last 3 steps in mitochondrial long-chain fatty acid oxidation. STUDY DESIGN: We performed molecular prenatal diagnosis in 9 pregnancies, 8 in 6 families with isolated long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency and one in a family with complete TFP deficiency. Analyses were performed on chorionic villous samples in 7 pregnancies and on amniocytes in 2. RESULTS: Molecular prenatal diagnosis successfully identified the fetal genotype in all 9 pregnancies. Two fetuses were affected, and both pregnancies were terminated by family decision. Two other fetuses had normal genotype and 5 others were heterozygotes. These 7 pregnancies were uncomplicated, and all the offspring are alive and apparently healthy. Genotypes of the aborted fetuses and neonates were confirmed by molecular analysis and enzymatic assays. CONCLUSIONS: Molecular prenatal diagnosis is possible and valid in guiding management of pregnancies in families with known TFP defects. Women heterozygous for TFP alpha-subunit mutations who carry fetuses with wild-type or heterozygous genotypes have uncomplicated pregnancies.

Enzymatic and molecular biological analysis of palmitoyl protein thioesterase deficiency in infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a neurodegenerative lysosomal storage disease that results from a deficiency of palmitoyl protein thioesterase (PPT), a deacylating enzyme that removes cysteine-bound palmitate from proteins. We developed an in vitro PPT enzyme assay that can be readily used for the clinical diagnosis of both INCL patients and carriers. The substrate is a palmitoylated peptide (IRY[14C]palmitoyl-CWLRR) synthesized by reacting [14C] palmitoyl-CoA with a synthetic octapeptide from the PNS P0 glycoprotein. The PPT assay performed in immortalized lymphoblastoid B-cells or the postmortem brain homogenate showed the optimal enzyme activity at pH 5.0, consistent with the findings that PPT is a lysosomal enzyme. PPT activity in lymphoblasts from INCL patients was <4% of that of control lymphoblasts. In addition, obligatory carriers showed 74% of the control activity. Other pathological controls, including the juvenile form of NCL, showed PPT activities that were not different from normal. Furthermore, one brain sample from an INCL patient contained only 7% PPT activity when compared with an unaffected brain. Thus, the enzyme activity assay gives a confident diagnosis of INCL. In contrast, when the total RNA extract from lymphoblasts was probed with 32P-labeled PPT by northern blot analysis, the level of transcript varied among independent INCL families and was not related to PPT activity. In conclusion, whereas variant genetic modifications result in PPT deficiency, all giving similar INCL phenotype, both affected patients and heterozygote carriers can now be screened with a reliable in vitro PPT assay.

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.

Palmitoyl-protein thioesterase and the molecular pathogenesis of infantile neuronal ceroid lipofuscinosis.

Palmitoyl-protein thioesterase (PPT) has recently been shown to be the defective enzyme underlying the infantile form of neuronal ceroid lipofuscinosis (INCL). In this paper, we review the enzymology of PPT, evidence for its localization in lysosomes, and recent advances in understanding the metabolic defect caused by PPT deficiency. Absence of PPT activity in lysosomes isolated from INCL lymphoblasts is demonstrated. A model for the formation of the storage bodies in INCL involving defective autophagocytic proteolysis is proposed.

Low molecular weight storage material in infantile ceroid lipofuscinosis (CLN1).

The identification of the genetic defect in CLN1 as a palmitoyl-protein thioesterase deficiency initiated a search for the lysosomal storage material. Pulse-chase labelling of fibroblasts and lymphoblastoid cell lines with [35S]cysteine revealed the presence of lipid [35S]cysteine material in CLN1 fibroblasts and not in controls, CLN2 or CLN3 patients or other patients with lipidosis. A single band comigrated with the acylcysteine standard and labelling with [3H]palmitate showed a band of material which eluted from the silicic acid column with the phospholipid fraction and which co-migrated with the lipid-[35S]cysteine band. The storage material is tentatively identified as palmitoylcysteine.