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Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT-PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179 bp of coding sequence and a premature stop codon. A repaired cDNA was constructed abolishing the splice site without changing the amino acid sequence. The level of glucocerebrosidase expression was increased sixfold. These data demonstrate that for maximum expression of any cDNA construct, the transcription products should be examined.

Background: Carnitine is essential for fatty acid metabolism. Free carnitine (FCA) is excreted in the urine in the glomerulus, but is partly reabsorbed by a carnitine transporter. The mechanism underlying the decrease in serum carnitine level during pregnancy is unclear. Objective: To investigate whether low carnitine level is associated with increased renal excretion in pregnant women. Methods: We recruited 43 healthy pregnant and 25 non-pregnant women. Total carnitine (TCA) and FCA levels were measured using the enzymatic cycling method, and the acylcarnitine (ACA) level was calculated. Fractional excretion (FE) was calculated as carnitine clearance divided by creatinine clearance. Results: The mean TCA, FCA, and ACA levels were lower at 12 weeks of gestation in pregnant than non-pregnant women (P <.001); the levels decreased further at 36 weeks, reaching 39%, 36%, and 52% of those in non-pregnant women, respectively (P <.001). The FEs were 3–4-fold higher in pregnant women than non-pregnant women. Pregnant women had a lower serum FCA/TCA ratio than non-pregnant women (0.788 ± 0.098 vs 0.830 ± 0.074, respectively; P <.05), whereas the urine FCA/TCA ratio was similar between the groups. Conclusion: Low carnitine level is associated with increased renal excretion during late pregnancy. [ABSTRACT FROM AUTHOR]

Carnitine is an essential cofactor for long-chain fatty acid oxidation. We characterized the human carnitine transporter in vitro in a cultured skin fibroblast model both at the previously established Km concentration of carnitine uptake in fibroblasts (5 mumol/liter) and at 0.05% Km (0.25 mumol/liter). A rapid exponential dose-dependent decrease in mean percentage of carnitine uptake was demonstrated with increasing concentrations of nigericin, but no significant decrease was found with equimolar amounts of valinomycin. This would suggest that the Na+ gradient is integral to carnitine transport function. Interference of the Na+ (out-in) gradient by nigericin may be secondary to cytoplasmic acidification by this K+ proton ionophore. The rate of uptake was fully saturated at an extracellular Na+ concentration of 150 mmol/liter. Replacement of 150 mmol/liter extracellular Na+ with Li+ resulted in an 80 and a 50% reduction, and replacement with K+ and Rb+ ions resulted in a 100 and an 85 to 90% reduction in carnitine uptake, respectively, at carnitine concentrations of 0.25 and 5 mumol/liter, underlining the specific requirement for the Na+ ion. The effects of different site-specific respiratory chain toxins, namely, rotenone (complex I), antimycin A (complex III), and potassium cyanide (KCN) (complex IV) on carnitine uptake was also examined. A rapid exponential dose-dependent decrease in mean percentage of carnitine uptake with increasing concentrations of inhibitors was demonstrated. These data suggest either a metabolic energy requirement of the carnitine transporter or interference of the Na+ (out-in) gradient by a proton gradient (in-out) secondary to the accumulation of intracellular H+ ions, due to the action of the respiratory chain toxins, further suggesting that the transporter is sensitive to and inhibited by intracellular H+ ions. The effects of several sulfhydryl-binding agents, namely 2,4-dinitrofluorobenzene, N-ethylmaleimide, and mersalyl acid, were examined, and a significant inhibition of carnitine uptake was demonstrated, suggesting that free sulfhydryl groups are also integral to the import function of the human fibroblast transporter.

Five metabolites of syn-3-ethyl-7-methyl-3,7-diazabicyclo[3.3.1]non-9-yl 4-chlorobenzoate hydrochloride (YUTAC) (1) were prepared and examined for Na+ current blocking activity in guinea pig ventricular myocytes. These metabolites showed lower inhibitory activities than the parent compound or were inactive.

Purpose: This study aimed to develop a novel exfoliating material with high efficacy and low irritation by synthesizing the Mandelic acid_Carnitine ion pairing complex (M_C complex) and evaluating its exfoliating properties. Additionally, the study assessed the skin improvement effects of the M_C complex through clinical evaluations. Methods: The M_C complex was synthesized in a 1:1 molar ratio of Mandelic acid and Carnitine. Structural characterization was performed using dynamic light scattering and Fourier‐transform infrared spectroscopy. Exfoliating efficacy was evaluated on porcine skin, and clinical assessments were conducted on human subjects to measure various skin improvement parameters. Results: The formation of the M_C complex was confirmed through particle size analysis, zeta‐potential measurements, and FT‐IR spectroscopy. The M_C complex demonstrated superior exfoliating efficacy compared to Mandelic acid alone, especially at pH 4.5. Clinical evaluations showed significant improvements in blackheads, whiteheads, pore volume, depth, density, count, and affected area, as well as skin texture. No adverse reactions were observed. Conclusion: The M_C complex exhibits high exfoliating efficacy and minimal irritation, making it a promising cosmetic ingredient for improving skin health. These findings support its potential as a low‐irritation exfoliating material under mildly acidic conditions, contributing to overall skin health enhancement. [ABSTRACT FROM AUTHOR]

Tay‒Sachs disease (TSD), a deficiency of β-hexosaminidase A (Hex A), is a rare but debilitating hereditary metabolic disorder. Symptoms include extensive neurodegeneration and often result in death in infancy. We report an in silico study of 42 Hex A variants associated with the disease. Variants were separated into three groups according to the age of onset: infantile (n=28), juvenile (n=9) and adult (n=5). Protein stability, aggregation potential and the degree of conservation of residues were predicted using a range of in silico tools. We explored the relationship between these properties and the age of onset of TSD. There was no significant relationship between protein stability and disease severity or between protein aggregation and disease severity. Infantile TSD had a significantly higher mean conservation score than nondisease associated variants. This was not seen in either juvenile or adult TSD. This study has established that the degree of residue conservation may be predictive of infantile TSD. It is possible that these more highly conserved residues are involved in trafficking of the protein to the lysosome. In addition, we developed and validated software tools to automate the process of in silico analysis of proteins involved in inherited metabolic diseases. Further work is required to identify the function of well-conserved residues to establish an in silico predictive model of TSD severity. [ABSTRACT FROM AUTHOR]

This study tested the hypothesis that in human aging, a decreased intramuscular acylcarnitine status is associated with (pre-)frailty, reduced physical performance, and altered mitochondrial function. We used a cross-sectional study design with well-matched fit and (pre-)frail old males and females, using young males and females as healthy controls. Frailty was assessed according to the Fried criteria and physical performance was determined by 400 m walk test, short physical performance battery and handgrip strength. Muscle and plasma acylcarnitine status, and muscle mitochondrial gene expression was analyzed. Results showed that intramuscular total carnitine levels and short-chain acylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. The low intramuscular short-chain acylcarnitine levels in females correlated with low physical performance, even after correction for muscle mass (%), and were accompanied with lowered expression of genes involved in mitochondrial energy production and functionality. It is, therefore, concluded that in (pre-)frail old females, intramuscular total carnitine levels and short-chain acylcarnitine levels are decreased, and this decrease is associated with reduced physical performance and low expression of a wide range of genes critical for mitochondrial function. The results stress the importance of taking sex differences into account in aging research., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Sphingolipidoses are inherited genetic diseases due to mutations in genes encoding proteins involved in the lysosomal catabolism of sphingolipids. Despite a low incidence of each individual disease, altogether, the number of patients involved is relatively high and resolutive approaches for treatment are still lacking. The chaperone therapy is one of the latest pharmacological approaches to these storage diseases. This therapy allows the mutated protein to escape its natural removal and to increase its quantity in lysosomes, thus partially restoring the metabolic functions. Sandhoff disease is an autosomal recessive inherited disorder resulting from β-hexosaminidase deficiency and characterized by large accumulation of GM2 ganglioside in brain. No enzymatic replacement therapy is currently available, and the use of inhibitors of glycosphingolipid biosynthesis for substrate reduction therapy, although very promising, is associated with serious side effects. The chaperone pyrimethamine has been proposed as a very promising drug in those cases characterized by a residual enzyme activity. In this review, we report the effect of pyrimethamine on the recovery of β-hexosaminidase activity in cultured fibroblasts from Sandhoff patients. [ABSTRACT FROM AUTHOR]

Objective Hereditary spastic paraplegias ( HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified ( SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism. Methods We describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes. Results Fibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ ZFYVE26 and the SPG11 protein spatacsin were interdependent. Interpretation Emerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work with Zfyve26−/− mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction. [ABSTRACT FROM AUTHOR]

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside. [ABSTRACT FROM AUTHOR]

Background: The two types of skin aging (intrinsic and extrinsic) share important molecular features, while having distinct differences on the macromolecular level: both lead to increased production of reactive oxygen species, enhanced matrix metalloproteinase expression and decreased procollagen synthesis. L-Carnitine L-tartrate has been reported to have anti-aging effects. Aim and Methods: The delivery as well as the transport of L-carnitine to the target sites, i.e. stratum corneum and epidermis-dermis of female abdominal skin, with and without the use of Pheroid™ as delivery system, were investigated during this study by use of Franz diffusion cells and analysed by liquid chromatography/mass spectrometry. The presence of endogenous L-carnitine in human skin was also investigated. Results and Conclusion: The Pheroid™ delivery system enhanced the diffusion of L-carnitine through the skin, although the phosphate buffer solution (PBS) showed a higher concentration of the active agent in the skin layers. This could be because Pheroid, being more lipophilic than PBS, improved the diffusion of L-carnitine through the skin layers, consequently leading to a lesser amount of L-carnitine delivered to the target site, i.e. the epidermis-dermis. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 2.3.1.78) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small aminoterminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as Nacetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction. [ABSTRACT FROM AUTHOR]

AbstractMany human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range ofin vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successfulin vivo use and development of PCs to treat LSDs will be discussed. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the anti-obesity effect of a mixture composed of Garcinia cambogia extract, soypeptide, and l-carnitine (1.2:0.3:0.02, w/w/w) in rats rendered obese by a high-fat diet (HFD). Sprague-Dawley rats were fed either the high-fat control diet (CD) or the 0.38% mixture-supplemented HFD (CD + M) for 9 weeks. The mixture significantly reduced body weight gain and the accumulation of visceral fat mass in a rat model of HFD-induced obesity. Moreover, the mixture effectively lowered blood and hepatic lipid concentrations and serum glucose, insulin, c-peptide, and leptin levels in rats with HFD-induced obesity. Results from real-time reverse transcription-polymerase chain reaction analyses indicated that the expression levels of leptin, tumor necrosis factor-alpha (TNF-α), and sterol regulatory element binding protein 1c (SREBP1c) genes in the epididymal fat tissue of rats fed the CD + M diet were 0.4-, 0.6-, and 0.48-fold, respectively, of those found in the CD rats ( P < 0.05), while expression of the uncoupling protein 2 (UCP2) gene in epididymal adipose tissue was 1.25-fold ( P < 0.05) of that found in CD rats. In conclusion, a mixture composed of G. cambogia extract, soy peptide, and l-carnitine attenuated visceral fat accumulation and improved dyslipidemia in a rat model with HFD-induced obesity. [ABSTRACT FROM AUTHOR]

L-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of L-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of L-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of L-[³H]carnitine uptake were investigated with or without specific inhibitors. L-Carnitine uptake in mature cells was sodium dependent and linear with time. K[sub m] and V[sub max] values for saturable uptake were 14.07 ± 1.70 µM and 26.3 ± 0.80 pmol·mg protein[sup -1]·6 min[sup -1], respectively. L-carnitine uptake was inhibited (P < 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that L-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM. [ABSTRACT FROM AUTHOR]

Summary: Early-onset dilatative and/or hypertrophic cardiomyopathy with episodic hypoglycaemic coma and very low serum and tissue concentrations of carnitine should alert the clinician to the probability of the plasmalemmal high-affinity carnitine transporter defect. The diagnosis can be established by demonstration of impaired carnitine uptake in cultured skin fibroblasts or lymphoblasts and confirmed by mutation analysis of the human OCTN2 gene in the affected child and obligate heterozygote parents. The institution of high-dose oral carnitine supplementation reverses the pathology in this otherwise lethal autosomal recessive disease of childhood, and carnitine therapy from birth in prospectively screened siblings may altogether prevent the development of the clinical phenotype. Heterozygotes may be at risk for cardiomyopathy in later adult life, particularly in the presence of additional risk factors such as hypertension and competitive pharmacological agents. OCTN2 belongs to a family of organic cation/carnitine transporters that function primarily in the elimination of cationic drugs and other xenobiotics in kidney, intestine, liver and placenta. The high- and low-affinity human carnitine transporters, OCTN2 and OCTN1, are multifunctional polyspecific organic cation transporters; therefore, defects in these transporters may have widespread implications for the absorption and/or elimination of a number of key pharmacological agents such as cephalosporins, verapamil, quinidine and valproic acid. A third organic/cation carnitine transporter with high specificity for carnitine, Octn3, has been clonedin mice. The juvenile visceral steatosis (jvs) mouse serves as an excellent clinical, biochemical and molecular model for the high-affinity carnitine transporter OCTN2 defect and is due to a spontaneous point mutation in the murine Octn2 gene on mouse chromosome 11, which is syntenic to the human locus at 5q31 that harbours the human OCTN2 gene. [ABSTRACT FROM AUTHOR]

Focuses on the known genetic defects of cellular carnitine transport and on the enzymes of the mitochondrial carnitine-dependent fatty acid entry pathway. Biosynthesis of carnitine; Homeostasis of body carnitine; Information on acylcarnitines.