Leucine Supplementation: A Novel Strategy for Modulating Lipid Metabolism and Energy Homeostasis.

Lipid metabolism is an important and complex biochemical process involved in the storage of energy and maintenance of normal biological functions. Leucine, a branched amino acid, has anti-obesity effects on glucose tolerance, lipid metabolism, and insulin sensitivity. Leucine also modulates mitochondrial dysfunction, representing a new strategy to target aging, neurodegenerative disease, obesity, diabetes, and cardiovascular disease. Although various studies have been carried out, much uncertainty still exists and further studies are required to fully elucidate the relationship between leucine and lipid metabolism. This review offers an up-to-date report on leucine, as key roles in both lipid metabolism and energy homeostasis in vivo and in vitro by acceleration of fatty acid oxidation, lipolysis, activation of the adenosine 5'-monophosphate-activated protein kinase (AMPK)-silent information regulator of transcription 1 (SIRT1)-proliferator-activated receptor γ coactivator-1α (PGC-1α) pathway, synthesis, and/or secretion of adipokines and stability of the gut microbiota.

Functional investigation of an universally conserved leucine residue in subunit a of ATP synthase targeted by the pathogenic m.9176 T>G mutation.

Protons are transported from the mitochondrial matrix to the intermembrane space of mitochondria during the transfer of electrons to oxygen and shuttled back to the matrix by the a subunit and a ring of identical c subunits across the membrane domain (FO) of ATP synthase, which is coupled to ATP synthesis. A mutation (m.9176 T > G) of the mitochondrial ATP6 gene that replaces an universally conserved leucine residue into arginine at amino acid position 217 of human subunit a (aL217R) has been associated to NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) and MILS (Maternally Inherited Leigh's Syndrome) diseases. We previously showed that an equivalent thereof in Saccharomyces cerevisiae (aL237R) severely impairs subunit a assembly/stability and decreases by >90% the rate of mitochondrial ATP synthesis. Herein we identified three spontaneous first-site intragenic suppressors (aR237M, aR237T and aR237S) that fully restore ATP synthase assembly. However, mitochondrial ATP synthesis rate was only partially recovered (40-50% vs wild type yeast). In light of recently described high-resolution yeast ATP synthase structures, the detrimental consequences of the aL237R change can be explained by steric and electrostatic hindrance with the universally conserved subunit a arginine residue (aR176) that is essential to FO activity. aL237 together with three other nearby hydrophobic residues have been proposed to prevent ion shortage between two physically separated hydrophilic pockets within the FO. Our results suggest that aL237 favors subunit c-ring rotation by optimizing electrostatic interaction between aR176 and an acidic residue in subunit c (cE59) known to be essential also to the activity of FO.

Leucine alters immunoglobulin a secretion and inflammatory cytokine expression induced by lipopolysaccharide via the nuclear factor-κB pathway in intestine of chicken embryos.

The mammalian target of rapamycin (mTOR) has been shown to be involved in lipopolysaccharide (LPS)-induced immune responses in many mammal cells. Here, we suggest that the mTOR pathway is involved in the intestinal inflammatory responses evoked by LPS treatment in chicken embryos. The intestinal tissue from Specific pathogen free chick embryos was cultured in the presence of LPS for 2 h. Secretory immunoglobulin A (sIgA) concentrations, messenger RNA (mRNA) expression of cytokines, and protein levels of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), mTOR and p70 ribosomal S6 kinase (p70S6K) were determined. The results showed that LPS treatment increased sIgA concentrations in a dose-dependent manner. The mRNA levels of interleukine (IL)-6, IL-8, IL-10, tumor necrosis factor-α and Toll-like receptor (TLR) 4 were upregulated by LPS treatment (P<0.05). Lipopolysaccharide increased the phosphorylation of Jun N-terminal kinase (JNK), p38 MAPK and NF-κB (P<0.05) while decreasing the phosphorylation level of mTOR (P<0.05). Supplementation of leucine at doses of 10, 20 and 40 mM dose-dependently decreased sIgA production. Leucine supplementation at 40 mM restored the phosphorylation level of mTOR and p70S6K while suppressing the phosphorylation levels of NF-κB (P<0.05) and partially down-regulating the phosphorylation of p38 MAPK and JNK. The transcription of IL-6 was significantly decreased by leucine supplementation. These results suggested that leucine could alleviate LPS-induced inflammatory responses by down-regulating NF-κB signaling pathway and evoking mTOR/p70S6K signaling pathway, which may involve in the regulation of the intestinal immune system in chicken embryos.

The serine protease, dipeptidyl peptidase IV as a myokine: dietary protein and exercise mimetics as a stimulus for transcription and release.

Dipeptidyl-peptidase IV (DPP-IV) is an enzyme with numerous roles within the body, mostly related to regulating energy metabolism. DPP-IV is also a myokine, but the stimulus for its release is poorly understood. We investigated the transcription and release of DPP-IV from skeletal muscle in a three-part study using C2C12 myotube cultures, an acute rat exercise and postexercise feeding model, and human feeding or human exercise models. When myotubes were presented with leucine only, hydrolyzed whey protein, or chemicals that cause exercise-related signaling to occur in cell culture, all caused an increase in the mRNA expression of DPP-IV (1.63 to 18.56 fold change, P < 0.05), but only whey protein caused a significant increase in DPP-IV activity in the cell culture media. When rats were fed whey protein concentrate immediately following stimulated muscle contractions, DPP-IV mRNA in both the exercised and nonexercised gastrocnemius muscles significantly increased 2.5- to 3.7-fold (P < 0.05) 3-6 h following the exercise/feeding bout; of note exercise alone or postexercise leucine-only feeding had no significant effect. In humans, plasma and serum DPP-IV activities were not altered by the ingestion of whey protein up to 1 h post consumption, after a 10 min bout of vigorous running, or during the completion of three repeated lower body resistance exercise bouts. Our cell culture and rodent data suggest that whey protein increases DPP-IV mRNA expression and secretion from muscle cells. However, our human data suggest that DPP-IV is not elevated in the bloodstream following acute whey protein ingestion or exercise.

Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons.

In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA-eEF1A-GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome.

A bifunctional protein regulates mitochondrial protein synthesis.

Mitochondrial gene expression is predominantly regulated at the post-transcriptional level and mitochondrial ribonucleic acid (RNA)-binding proteins play a key role in RNA metabolism and protein synthesis. The AU-binding homolog of enoyl-coenzyme A (CoA) hydratase (AUH) is a bifunctional protein with RNA-binding activity and a role in leucine catabolism. AUH has a mitochondrial targeting sequence, however, its role in mitochondrial function has not been investigated. Here, we found that AUH localizes to the inner mitochondrial membrane and matrix where it associates with mitochondrial ribosomes and regulates protein synthesis. Decrease or overexpression of the AUH protein in cells causes defects in mitochondrial translation that lead to changes in mitochondrial morphology, decreased mitochondrial RNA stability, biogenesis and respiratory function. Because of its role in leucine metabolism, we investigated the importance of the catalytic activity of AUH and found that it affects the regulation of mitochondrial translation and biogenesis in response to leucine.

Arginine 199 and leucine 208 have key roles in the control of adenosine A2A receptor signalling function.

One successful approach to obtaining high-resolution crystal structures of G-protein coupled receptors is the introduction of thermostabilising mutations within the receptor. This technique allows the generation of receptor constructs stabilised into different conformations suitable for structural studies. Previously, we functionally characterised a number of mutants of the adenosine A2A receptor, thermostabilised either in an agonist or antagonist conformation, using a yeast cell growth assay and demonstrated that there is a correlation between thermostability and loss of constitutive activity. Here we report the functional characterisation of 30 mutants intermediate between the Rag23 (agonist conformation mutant) and the wild-type receptor using the same yeast signalling assay with the aim of gaining greater insight into the role individual amino acids have in receptor function. The data showed that R199 and L208 have important roles in receptor function; substituting either of these residues for alanine abolishes constitutive activity. In addition, the R199A mutation markedly reduces receptor potency while L208A reduces receptor efficacy. A184L and L272A mutations also reduce constitutive activity and potency although to a lesser extent than the R199A and L208A. In contrast, the F79A mutation increases constitutive activity, potency and efficacy of the receptor. These findings shed new light on the role individual residues have on stability of the receptor and also provide some clues as to the regions of the protein responsible for constitutive activity. Furthermore, the available adenosine A2A receptor structures have allowed us to put our findings into a structural context.

Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine.

Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.

L-leucine, L-methionine, and L-lysine are involved in the regulation of intermediary metabolism-related gene expression in rainbow trout hepatocytes.

Using rainbow trout hepatocytes stimulated with l-leucine, l-methionine, or l-lysine in the presence or absence of bovine insulin, we investigated the ability of these amino acids to mimic the effects of a pool of amino acids on protein kinase B (Akt)/target of rapamycin (TOR) signaling pathways and expression of 6 genes known to be subjected to insulin and/or amino acid regulation [glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GK), pyruvate kinase (PK), fatty acid synthase (FAS), and serine dehydratase (SDH)]. Emphasis was placed on leucine, known to be a signaling molecule in mammals, and methionine and lysine that are essential amino acids limiting in plant-based diets for fish. In the presence of insulin, leucine (but not methionine or lysine) phosphorylated Akt and ribosomal protein S6 as previously observed with a pool of amino acids, suggesting that leucine might participate in the activation of the TOR pathway by amino acids in fish, as in mammals. G6Pase, PEPCK, GK, and SDH gene expression were higher in leucine-treated cells compared with control cells. Leucine combined with insulin reduced G6Pase gene expression by 90% and increased FAS gene expression > 4-fold compared with the control treatment. Methionine weakly decreased G6Pase, GK, and SDH gene expression and lysine weakly but significantly decreased the mRNA level of PEPCK. Thus, leucine regulated gluconeogenesis and lipogenesis, but not glycolysis, in the same way as a pool of amino acids. Methionine appeared to be involved in the regulation of specific genes, whereas lysine only had limited effects. These findings are particularly relevant regarding the involvement of amino acids in the regulation of metabolism-related gene expression.

Two dileucine motifs mediate late endosomal/lysosomal targeting of transmembrane protein 192 (TMEM192) and a C-terminal cysteine residue is responsible for disulfide bond formation in TMEM192 homodimers.

TMEM192 (transmembrane protein 192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and an unknown function that was initially discovered by organellar proteomics. Subsequently, localization in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192, and homodimers of TMEM192 linked by disulfide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimaeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL-type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucine motifs resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulfide bond formation, mutagenesis of cysteine residues was performed. Mutation of Cys266 abolished disulfide bridge formation between TMEM192 molecules, indicating that TMEM192 dimers are linked by a disulfide bridge between their C-terminal tails. According to the predicted topology, Cys266 would be localized in the reductive milieu of the cytosol where disulfide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays, the localization of the N- and C-termini of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly.

Leucine at position 383 of fusion protein is responsible for fusogenicity of wild-type mumps virus in B95a cells.

OBJECTIVE: Mumps virus is isolated in Vero cells and, recently, B95a cells have been reported to be susceptible to it. Currently circulating wild-type mumps virus strains (genotypes B, G, J and L) induced cytopathic effects in both Vero and B95a cells. On the other hand, the Hoshino vaccine strain (KO3) did not induce cytopathic effects in B95a cells. In this study, differences in fusion inducibility were investigated. METHODS: Nucleotide sequences of the fusion (F) and hemagglutinin-neuraminidase (HN) protein regions were compared. The F and HN expression plasmids were constructed and fusion analysis was conducted, using recombinant F expression plasmids under the control of T7 RNA polymerase. RESULTS: Extensive cell fusion was observed when B95a cells were transfected with the wild-type F expression plasmid as the F expression partner; 13-16 amino acid differences were observed in the F protein region between the KO3 and the wild types. F expression plasmids with leucine at position 383 of the F protein induced large cell fusion in B95a cells. CONCLUSION: Leucine at position 383 of the F protein of the wild types was the critical amino acid for fusion inducibility in B95a cells.

Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity.

The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F(1). Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.

Deletion of v7-3 (SLC6A15) transporter allows assessment of its roles in synaptosomal proline uptake, leucine uptake and behaviors.

V7-3 (SLC6A15) is the prototype for a gene subfamily whose members have sequence homologies to classical Na+- and Cl(-)-dependent neurotransmitter transporters but display unusual features that include characteristic large fourth extracellular loops. Interest in v7-3 has been increased by the elucidation of its expression in neurons located in cerebral cortex, hippocampus, cerebellum, midbrain and olfactory bulb. To help clarify the role of v7-3 in brain functions, we have created and characterized v7-3 knockout mice. These mice lack functional v7-3 protein but are viable and fertile. While our studies were in progress, v7-3 expression was reported to confer transport of proline and branched-chain amino acids in in vitro expression systems [Takanaga, H., Mackenzie, B., Peng, J.B., Hediger, M.A., 2005b. Characterization of a branched-chain amino-acid transporter SBAT1 (SLC6A15) that is expressed in human brain. Biochem. Biophys. Res. Commun. 337, 892-900; Broer, A., Tietze, N., Kowalczuk, S., Chubb, S., Munzinger, M., Bak, L.K., Broer, S., 2006. The orphan transporter v7-3 (slc6a15) is a Na+-dependent neutral amino acid transporter (B0AT2). Biochem. J. 393, 421-430]. Assessment of amino acid uptake into cortical synaptosomes of v7-3 knockouts identified 15% and 40% reductions in sodium-dependent proline and leucine transport, respectively, compared to wild type controls. Despite these biochemical changes, v7-3 knockout mice demonstrate only modest alterations in rotarod performance with aging and lack reproducible alterations in other motor, memory, anxiety or olfactory tests. Compensation for the lack of v7-3 via other amino acid carriers is likely to leave v7-3 knockouts without gross behavioral manifestations. The current results place v7-3 in the context of other brain transporters that accumulate proline and branched-chain amino acids.

Enterococcal leucine-rich repeat-containing protein involved in virulence and host inflammatory response.

Enterococcus faecalis is an important nosocomial pathogen associated with high morbidity and mortality for patients who are immunocompromised or who have severe underlying diseases. The E. faecalis genome encodes numerous surface-exposed proteins that may be involved in virulence. This work describes the characterization of the first internalin-like protein in E. faecalis, ElrA, belonging to the recently identified WxL family of surface proteins. ElrA contains an N-terminal signal peptide for export, a leucine-rich repeat domain that may interact with host cells, and a C-terminal WxL domain that interacts with the peptidoglycan. Disruption of the elrA gene significantly attenuates bacterial virulence in a mouse peritonitis model. The elrA deletion mutant also displays a defect in infection of host macrophages and a decreased interleukin-6 response in vivo. Finally, elrA expression is induced in vivo. Altogether, these results demonstrate a role for ElrA in the E. faecalis infectious process in vivo and suggest that this surface protein may contribute to E. faecalis virulence by stimulating the host inflammatory response.

Specific mutations in transmembrane helix 8 of human concentrative Na+/nucleoside cotransporter hCNT1 affect permeant selectivity and cation coupling.

The Na+/nucleoside cotransporters hCNT1 (650 residues) and hCNT2 (658 residues) are 72% identical in amino acid sequence and contain 13 putative transmembrane helices (TMs). Both transport uridine and adenosine but are otherwise selective for pyrimidine (system cit) and purine (system cif) nucleosides, respectively. Previously, we used site-directed mutagenesis and functional expression in Xenopus oocytes to identify two pairs of adjacent residues in TMs 7 and 8 of hCNT1 (Ser319-Gln320 and Ser353-Leu354) that, when converted to the corresponding residues in hCNT2 (Gly-Met and Thr-Val, respectively), changed the permeant selectivity of the transporter from cit to cif. We now report an investigation of the effects of corresponding mutations in TM 8 alone and demonstrate unique S353T- and L354V-induced changes in nucleoside specificity and cation coupling, respectively. hCNT1 mutation S353T produced a profound decrease in cytidine transport efficiency (Vmax/Km ratio) and, in combination with L354V (S353T/L354V), resulted in a novel uridine-preferring transport phenotype. In addition, the L354V mutation markedly increased the apparent affinity of hCNT1 for Na+ and Li+. Both hCNT1 TM 8 residues exhibited uridine-protectable inhibition by p-chloromercuribenzene sulfonate when converted to Cys, suggesting that they occupy positions within or closely adjacent to a common cation/nucleoside translocation pore.

Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting.

The 5-HT1A and 5-HT1B serotonin receptors exhibit different subcellular localizations in neurons. Evidence has been reported that the C-terminal domain is involved in the somato-dendritic and axonal targeting of 5-HT1AR and 5-HT1BR, respectively. Here we analyzed the consequences of the mutation of a di-leucine motif and palmitoylated cysteines within this domain. Replacement of I414-I415 by a di-alanine in 5-HT1AR led to endoplasmic reticulum (ER) sequestration of the corresponding mutant expressed in cell lines as well as in hippocampal neurons in culture. Furthermore, di-leucine-mutated receptors were unable to bind 5-HT1A agonists and presented a major deficit in their glycosylation state, suggesting that they are misfolded. By contrast, mutation of the di-leucine motif in the C-terminal domain of 5-HT1BR had no major consequence on its subcellular targeting. However, in the case of the 1ActB chimera (substitution of the C-terminal domain of the 5-HT1BR into 5-HT1AR), this mutation was also found to cause sequestration within the ER. Replacement of palmitoylated cysteines by serines had no consequence on either receptor type. These data indicate that the di-leucine motif of the 5-HT1AR and 5-HT1BR tails is implicated in proper folding of these receptors, which is necessary for their ER export.

Regulation of protein synthesis by leucine starvation involves distinct mechanisms in mouse C2C12 myoblasts and myotubes.

Leucine modulates protein translation in higher eukaryotes by affecting phosphorylation and the function of proteins that regulate the initiation and/or elongation steps. These include the initiation factor 4E binding protein 1 (4E-BP1), initiation factor 4E (eIF4E), initiation factor 2 (eIF2alpha), ribosomal S6 kinases (S6K1/2), and elongation factor 2 (eEF2). The alteration of protein translation by leucine starvation was studied during myogenic differentiation using the mouse C2C12 cell line as well as the role of rapamycin-sensitive mTOR (mammalian target of rapamycin) in the signaling of leucine in myotubes. A time course study showed that 1 h of leucine starvation decreased protein synthesis and S6K1 phosphorylation in myoblasts, whereas 3-5 h of starvation were necessary to induce such an alteration in myotubes. Although S6K1 phosphorylation was reduced in leucine-deprived myotubes, S6K2 and S6 phosphorylation were not affected. In contrast, rapamycin decreased the phosphorylation of S6K2 and S6 in myotubes. It is therefore likely that under the conditions present, the rapamycin-sensitive mTOR was not affected by leucine starvation. S6K1 dephosphorylation may thus be mTOR independent, and the functional mTOR/S6K2 pathway may maintain S6 phosphorylation. An increased phosphorylation of eEF2 in myoblasts and myotubes indicated that global protein synthesis was reduced via a decrease in translation elongation. An increased association between 4E-BP1 and eIF4E, and increased phosphorylation of eIF2alpha also contributed to decreasing protein synthesis in leucine-starved myoblasts. In contrast, in leucine-starved myotubes, there were no change in the 4E-BP1-eIF4E association or eIF2alpha phosphorylation, suggesting that these factors were not rate limiting for decreasing protein synthesis in leucine-deprived myotubes.

Molecular mechanism of the constitutive activation of the L250Q human melanocortin-4 receptor polymorphism.

The Melanocortin-4 Receptor is a G-protein coupled receptor that has been physiologically linked to participate in the regulation of energy homeostasis. The Melanocortin-4 Receptor is stimulated by endogenous melanocortin agonists derived from the pro-opiomelanocortin gene transcript and antagonized by the endogenous antagonist agouti-related protein. Central administration of melanocortin agonists has been demonstrated to decrease food intake and conversely, treatment with antagonists resulted in increased food intake. Deletion of the Melanocortin-4 Receptor gene from the mouse genome results in an obese and hyperphagic phenotype. Polymorphisms of the human Melanocortin-4-Receptor have been found in severely obese individuals, suggesting that Melanocortin-4 Receptor malfunction might be involved in human obesity and obesity-associated diabetes. Herein, we have performed experiments to understand the molecular mechanisms associated with the L250Q human Melanocortin-4-Receptor polymorphism discovered in an extremely obese woman. This L250Q human Melanocortin-4-Receptor has been pharmacologically characterized to result in a constitutively active receptor. The fact that a constitutively active human Melanocortin-4-Receptor mutation was found in an obese person is a physiologic contradiction, as chronic activation of the human Melanocortin-4-Receptor and subsequently high cyclic adenosine monophosphate levels should theoretically result in a normal or lean phenotype. In this study, we demonstrated that agouti-related protein acts as an inverse agonist at this constitutively active receptor, and we propose a mechanism by which agouti-related protein might contribute to the obese phenotype in the L250Q patient. In addition, using receptor mutagenesis, pharmacology, and computer modeling approaches, we investigated the molecular mechanism by which modification of the L250 residue results in constitutive activation of the human Melanocortin-4-Receptor.

Endocytosis of the Nipah virus glycoproteins.

Nipah virus (NiV), a highly pathogenic member of the family Paramyxoviridae, encodes the surface glycoproteins F and G. Since internalization of the NiV envelope proteins from the cell surface might be of functional importance for viral pathogenesis either by regulating cytopathogenicity or by modulating recognition of infected cells by the immune system, we analyzed the endocytosis of the NiV F and G proteins. Interestingly, we found both glycoproteins to be internalized in infected and transfected cells. As endocytosis is normally mediated by tyrosine- or dileucine-dependent signals in the cytoplasmic tails of transmembrane proteins, all potential internalization signals in the NiV glycoproteins were mutated. Whereas the G protein appeared to be constitutively internalized with the bulk flow during membrane turnover, uptake of the F protein was found to be signal mediated. F endocytosis clearly depended on a membrane-proximal YXXPhi motif and was found to be of functional importance for the biological activity of the protein.

Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity.

HIF (hypoxia-inducible factor) is an alphabeta transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-alpha are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-alpha (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340-->Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344-->Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.