Large neutral amino acid levels tune perinatal neuronal excitability and survival.

Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.

Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation.

A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis.

Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals.

5-Aminovalerate (5AVA) is the precursor of valerolactam, a potential building block for producing nylon 5, and is a C5 platform chemical for synthesizing 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putidadavAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured in a medium containing 20g/L of glucose and 10g/L of L-lysine, 3.6g/L of 5AVA was produced by converting 7g/L of L-lysine. When the davAB genes were introduced into recombinant E. coli strainXQ56allowing enhanced L-lysine synthesis, 0.27 and 0.5g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate could be demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 20g/L glucose, 10g/L L-lysine and 10g/L α-ketoglutarate, 1.7g/L of glutarate was produced.

Synaptic Vesicle Protein NTT4/XT1 (SLC6A17) Catalyzes Na+-coupled Neutral Amino Acid Transport.

The SLC6 family of structurally related, Na(+)-dependent transporter proteins is responsible for presynaptic reuptake of the majority of neurotransmitters. Within this family are a number of orphan transporters, including NTT4/XT1 (SLC6A17), a protein first identified over 15 years ago. NTT4/XT1 is expressed exclusively in the nervous system and specifically on synaptic vesicles in glutamatergic and some GABAergic neurons. Despite extensive efforts by a number of groups, no substrate has been reported for NTT4/XT1. Here we use a combination of molecular manipulations to increase expression of the NTT4/XT1 protein at the plasma membrane and to directly demonstrate that it catalyzes neutral amino acid transport. The substrate profile of the NTT4/XT1-dependent activity is similar to that of the closely related B(0)AT2/SBAT1 (SLC6A15), including a submillimolar apparent affinity for proline and leucine and a low millimolar apparent affinity for glutamine. The transport activity is Na(+)-dependent and Cl(-)-independent and is inhibited by low pH as is SLC6A15, suggesting redundant roles for these proteins. This characterization of NTT4/XT1 offers important insights into neurotransmitter metabolism as well as the mechanistic differences among the structurally related, but functionally divergent, SLC6 proteins.

Quercetin activates human Kv1.5 channels by a residue I502 in the S6 segment.

1. The aims of the present study were to investigate the pharmacological effects of quercetin on wild-type (WT) and mutant (I502A) human (h) Kv1.5 channel currents (I(kur)) and to identify whether mutation in the S6 segment is critical to activation of I(kur) by quercetin. 2. Experiments were performed on WT and site-directed mutant hKv1.5 channels, which were stably expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. 3. Quercetin increased WT hKv1.5 channel current in a concentration-, voltage- and time-dependent manner, with an EC(50) of 37.8 micromol/L and a negative shift in the steady state activation and inactivation curves. Quercetin accelerated channel activation and inactivation, significantly decreasing activation and inactivation time constants. However, mutating the I502 residue to Ala abolished the activating effect of quercetin. Quercetin did not modify the activation and inactivation kinetics of I502A channels. As an anti-oxidant, tanshinone IIA (4 micromol/L) inhibited the H(2)O(2)-induced activation of WT hKv1.5 channels. In contrast, quercetin had no significant effect. 4. We conclude that: (i) quercetin preferentially binds to and increases the current amplitude of WT hKv1.5 channels; (ii) Ile502, an aliphatic and neutral amino acid residue residing in the S6 segment, is important in quercetin binding; and (iii) quercetin-induced changes in the properties of WT hKv1.5 channels may be foreign to its own anti-oxidant action.

Prevalence of positive selection among nearly neutral amino acid replacements in Drosophila.

We have estimated the selective effects of amino acid replacements in natural populations by comparing levels of polymorphism in 91 genes in African populations of Drosophila melanogaster with their divergence from Drosophila simulans. The genes include about equal numbers whose level of expression in adults is greater in males, greater in females, or approximately equal in the sexes. Markov chain Monte Carlo methods were used to sample key parameters in the stationary distribution of polymorphism and divergence in a model in which the selective effect of each nonsynonymous mutation is regarded as a random sample from some underlying normal distribution whose mean may differ from one gene to the next. Our analysis suggests that approximately 95% of all nonsynonymous mutations that could contribute to polymorphism or divergence are deleterious, and that the average proportion of deleterious amino acid polymorphisms in samples is approximately 70%. On the other hand, approximately 95% of fixed differences between species are positively selected, although the scaled selection coefficient (N(e)s) is very small. We estimate that approximately 46% of amino acid replacements have N(e)s < 2, approximately 84% have N(e)s < 4, and approximately 99% have N(e)s < 7. Although positive selection among amino acid differences between species seems pervasive, most of the selective effects could be regarded as nearly neutral. There are significant differences in selection between sex-biased and unbiased genes, which relate primarily to the mean of the distributions of mutational effects and the fraction of slightly deleterious and weakly beneficial mutations that are fixed.

Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four Vlambda6 proteins.

Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (VL) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the VL domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable Vlambda6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic Vlambda6 protein (Wil) that had neutral amino acids at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of immunoglobulin light chains.