Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter

I. Background: E. coli is a widely used tool for the over-expression of human proteins for studying structure and function. The toxicity of human proteins for E. coli often hampers the expression. This study aims to find conditions for the expression of a membrane transporter known as the carnitine transporter CT2. The knowledge on this transporter is scarce, thus obtaining the recombinant protein is very important for further studies. II. Methods and Results: The cDNAs coding for human CT2 (hCT2) was cloned in the pH6EX3 vector and different transformed E. coli strains were cultured in absence or in presence of glucose. hCT2 expression was obtained. The protein was purified and reconstituted into proteoliposomes in a functionally active state.III. Conclusions: using the appropriate IPTG concentration, together with the addition of glucose, hCT2 has been expressed in E. coli. The protein is active and shows capacity to transport carnitine in proteoliposomes. The results have a great interest in basic biochemistry of membrane transporters and applications to human health since hCT2 is involved in human pathology.

Primary Carnitine Deficiency and Autism Spectrum Disorder is there a Relationship?

Carnitine plays essential role in energy metabolism .Systemic primary carnitine deficiency is a genetic disorder caused by decreased or absent organic cation transporter type 2 (OCTN2) carnitine transporter activity, resulting in low serum carnitine levels and decreased carnitine accumulation inside cells. The decrease carnitine results in impaired fatty acid oxidation. Primary carnitine deficiency presents a hypoketotic, hypoglycemia and hepatic encephalopathy. Recently, primary carnitine deficiency has been associated with neurodevelopmental disorders including autism spectrum disorders. A seven year-old schoolgirl with intellectual deficit, autistic features and primary carnitine deficiency has been reported. A significant decrease in carnitine levels has been shown in patients with autism and this has been related to the existence of a mitochondrial disease and more severe autism. The early identification of patients with low levels of carnitine or primary carnitine deficiency, with the different methods of measuring free carnitine, including tandem mass spectrometry could help to identify these patients early and achieve an early treatment and better neurological prognosis, because autism spectrum disorders may be preventable in this subgroup. We hope that this paper is useful to neurologists and pediatricians, and may give them more reason to suspect a diagnosis of PCD and autism.

Newborn Screening and Genetic Analysis Identify Six Novel Genetic Variants for Primary Carnitine Deficiency in Ningbo Area, China

Primary carnitine deficiency (PCD) is an autosomal recessive disorder that could result in sudden death. It is caused by a defect in the carnitine transporter encoded by SLC22A5 (Solute Carrier Family 22 Member 5, MIM:603377). Currently, a number of variants in SLC22A5 have been identified, however, the PCD prevalence and its variants in Ningbo area are unclear. In this study, we screened 265,524 newborns by using tandem mass spectrometry. Variants in SLC22A5 were further detected by next-generation sequencing in individuals with abnormal free carnitine levels (C0). We identified 53 newborns with abnormal C0 levels and 26 with variants in SLC22A5. Among them, 16 with compound heterozygous or homozygous variants in SLC22A5 were diagnosed with PCD, suggesting the PCD birth prevalence in Ningbo city was 1/16,595. Moreover, the C0 level was significantly (P = 0.013) higher in PCD patients than in those with one variant. Besides, the c.1400C > G (p. S467C) and c.51C > G (p. F17L) variants were the most frequent and six novel variants are all predicted to be damaging. This study reports the largest PCD patients in Ningbo area by newborn screening and expands the variant spectrum of SLC22A5. Our findings demonstrate the clinical value of combining NBS program results with DNA analysis for the diagnosis of PCD.

Fine mapping of a thrips resistance QTL in Capsicum and the role of diterpene glycosides in the underlying mechanism

Key message A major thrips resistance QTL in Capsicum was fine-mapped to a region of 0.4 Mbp, and a multidisciplinary approach has been used to study putative underlying mechanisms. Abstract Resistance to thrips is an important trait for pepper growers. These insects can cause extensive damage to fruits, flowers and leaves on field and greenhouse grown plants worldwide. Two independent studies in Capsicum identified diterpene glycosides as metabolites that are correlated with thrips resistance. In this study, we fine-mapped a previously defined thrips resistance QTL on chromosome 6, to a region of 0.4 Mbp harbouring 15 genes. Two of these 15 candidate genes showed differences in gene expression upon thrips induction, when comparing plants carrying the resistance allele in homozygous state to plants with the susceptibility allele in homozygous state for the QTL region. Three genes, including the two genes that showed difference in gene expression, contained a SNP that was predicted to lead to changes in protein structure. Therefore, these three genes, i.e. an acid phosphatase 1 (APS1), an organic cation/carnitine transporter 7 (OCT7) and an uncharacterized locus LOC107874801, are the most likely candidates for playing a role in thrips resistance and are a first step in elucidating the genetic basis of thrips resistance in Capsicum. In addition, we show that the diterpene glycoside profiles did not differ between plants with the resistance and susceptibility allele for the chromosome 6 QTL, suggesting that these compounds do not play a role in the resistance conferred by the genes located in the major thrips resistance QTL studied.

Organic Cation Transporters in Human Physiology, Pharmacology, and Toxicology

Individual cells and epithelia control the chemical exchange with the surrounding environment by the fine-tuned expression, localization, and function of an array of transmembrane proteins that dictate the selective permeability of the lipid bilayer to small molecules, as actual gatekeepers to the interface with the extracellular space. Among the variety of channels, transporters, and pumps that localize to cell membrane, organic cation transporters (OCTs) are considered to be extremely relevant in the transport across the plasma membrane of the majority of the endogenous substances and drugs that are positively charged near or at physiological pH. In humans, the following six organic cation transporters have been characterized in regards to their respective substrates, all belonging to the solute carrier 22 (SLC22) family: the organic cation transporters 1, 2, and 3 (OCT1–3); the organic cation/carnitine transporter novel 1 and 2 (OCTN1 and N2); and the organic cation transporter 6 (OCT6). OCTs are highly expressed on the plasma membrane of polarized epithelia, thus, playing a key role in intestinal absorption and renal reabsorption of nutrients (e.g., choline and carnitine), in the elimination of waste products (e.g., trimethylamine and trimethylamine N-oxide), and in the kinetic profile and therapeutic index of several drugs (e.g., metformin and platinum derivatives). As part of the Special Issue Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations, this article critically presents the physio-pathological, pharmacological, and toxicological roles of OCTs in the tissues in which they are primarily expressed.

Development of a novel molecular probe for the detection of liver mitochondrial redox metabolism

Redox status influences the course of the inflammatory, metabolic, and proliferative liver diseases. Oxidative stress is thought to play a crucial and sustained role in the pathological progression of early steatosis to severe hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Oxidative stress induced by reactive oxygen species which are generated in the mitochondria can lead to chronic organelle damage in hepatocytes. Currently, the diagnosis of liver disease requires liver biopsy, which is invasive and associated with complications. The present report describes the development of a novel molecular probe, EDA-PROXYL, with higher reactivity and mitochondrial selectivity than standard carboxyl-PROXYL and carbamoyl-PROXYL probes. The membrane permeability of our probe improved in aqueous environments which led to increased accumulation in the liver and interaction of EDA-PROXYL with the carnitine transporter via the amine (NH3+) group further increased accumulation. This increased mitochondrial sensitivity and enhanced accumulation highlight the potential of EDA-PROXYL as a molecular probe for determining metabolic reactions of the mitochondria. Thus, this novel probe could be a tool for the evaluation of redox status of the mitochondria to assess the degree of liver injury and, ultimately, the response to pharmacological therapy.

Metabolic Serendipities of Expanded Newborn Screening

Incidental findings on newborn screening (NBS) are results that are not the target of screening within a given NBS program, but rather are found as a result of the screening and resulting diagnostic workup for that target. These findings may not have an immediate clinical impact on the newborn, but are sometimes an additional benefit of NBS programs and may be considered secondary targets of NBS programs. This work describes four case reports that had incidental findings on the NBS, which eventually led to the diagnosis of another metabolic disease instead of the one that was initially suspected. The first case was a new defect in the cationic amino acid transporter-2 (CAT-2), which was oriented as an arginase-1 deficiency in the newborn. The second case was a maternal glutaric aciduria type 1 (GA-1) that mimicked a carnitine transporter deficiency in the newborn. The third report was a case of lysinuric protein intolerance (LPI), which appeared as high levels of citrulline on the NBS. The fourth case was a mother with homocystinuria that was diagnosed during the biochemical study of vitamin B12 status. All cases provide new or interesting data that will help guide differential diagnosis in the future.