Systematic Mutational Analysis of the Intracellular Regions of Yeast Gap1 Permease

The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g. ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER).Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

New pathways for the metabolism of fructosamines and other glycation products in bacteria

Glucose reacts spontaneously with amines of amino acids and proteins to form Schiff bases, which slowly undergo an Amadori rearrangement that converts them to fructosamines. This process, known as glycation, is observed with all types of reducing sugars. When glycation implicates fructose, the main rearrangement product, known as Heyns product, has a glucosamine-like structure. Before this thesis work was started, fructosamines were known to be metabolized in some bacteria and fungi by fructosyl-amino acid oxidases that most often convert fructosamines to glucosone and a free amine with production of H2O2. In mammals, fructosamine 3-kinase is known to catalyze the phosphorylation of fructosamines to fructosamine 3-phosphates, which spontaneously degrade to 3-deoxyglucosone, inorganic phosphate and a free amine. The seminal observation of the present thesis was the observation that Escherichia coli extracts catalyze the ATP-dependent phosphorylation of fructoselysine. This prompted us to study fructoselysine metabolism in this bacterium. We could show that this Amadori product supports the growth of E. coli, and that fructoselysine is phosphorylated in cell extracts to a compound that is converted to glucose 6-phosphate. The operon encoding the implicated enzymes was identified and given the designation frl. FrlA encodes a putative transporter. FrlD is a kinase of the PfkB family that was characterized and shown to phosphorylate fructoselysine to fructoselysine 6-phosphate. The latter is converted to glucose 6-phosphate and free lysine by fructoselysine-6-phosphate deglycase, the product of the frlB gene, which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. FrlC is homologous to tagatose 3-epimerase, which led us to hypothesize that it catalyzes the isomerization of fructoselysine and its C3 epimer, psicoselysine. This hypothesis was confirmed by overexpressing FrlC and characterizing its enzymatic activity. Furthermore, wild-type E. coli was found to grow on psicoselysine, but this ability (as well as that of growing on fructoselysine) was lost in two mutants of the frl operon, confirming the involvement of this operon in the metabolism of both lysine derivatives. A study of the Bacillus subtilis homologues of FrlD and FrlB, known as YurL and YurP, respectively, allowed us to demonstrate that they catalyze similar reactions as their E. coli counterpart, but that their specificity is totally different. Instead of acting on fructoselysine (or its 6-phospho-derivative), they preferentially act on fructosamine derivatives of hydrophobic a-amino acids. Finally, we noted that several bacterial operons that contain a putative fructosamine-6-phosphate deglycase also encode a second, closer homologue of the isomerase domain of glucosamine-6-phosphate synthase. Overexpression and characterization of these two proteins (encoded by an Enterococcus faecium operon) allowed us to show that the first one catalyzes the cleavage of fructoselysine 6-phosphate to glucose 6-phosphate and lysine and the second one, the cleavage of glucoselysine 6-phosphate to fructose 6-phosphate and lysine. Operons encoding this pair of enzymes also comprise a phosphotransferase system, which is presumably responsible for the transport and phosphorylation of both fructoselysine and glucoselysine. This new type of operon appears therefore to be responsible for the metabolism of both glycation and fructation products. In conclusion, our work has led to the identification of new enzymes involved in the metabolism of glycation products. These enzymes appear to be more common than fructosyl-amino acid oxidases in the bacterial world. Furthermore, they provide interesting tools for the assay and production of fructosamines and related compounds. ...Thèse de doctorat en sciences biomédicales (biochimie)(SBIM 3) -- UCL, 200

Fructoselysine 3-epimerase, an enzyme involved in the metabolism of the unusual Amadori compound psicoselysine in Escherichia coli.

The frl (fructoselysine) operon encodes fructoselysine 6-kinase and fructoselysine 6-phosphate deglycase, allowing the conversion of fructoselysine into glucose 6-phosphate and lysine. We now show that a third enzyme encoded by this operon catalyses the metal-dependent reversible interconversion of fructoselysine with its C-3 epimer, psicoselysine. The enzyme can be easily assayed through the formation of tritiated water from [3-3H]fructoselysine. Psicoselysine supports the growth of Escherichia coli, causing the induction of the three enzymes of the frl operon. No growth on fructoselysine or psicoselysine was observed with Tn5 mutants in which the putative transporter (FrlA) or fructoselysine 6-phosphate deglycase (FrlB) had been inactivated, indicating the importance of the frl operon for the metabolism of both substrates. The ability of E. coli to grow on psicoselysine suggests the occurrence of this unusual Amadori compound in Nature

Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli.

Escherichia coli was found to grow on fructoselysine as an energetic substrate at a rate of about one-third of that observed with glucose. Extracts of cells grown on fructoselysine catalyzed in the presence of ATP the phosphorylation of fructoselysine and a delayed formation of glucose 6-phosphate from this substrate. Data base searches allowed us to identify an operon containing a putative kinase (YhfQ) belonging to the PfkB/ ribokinase family, a putative deglycase (YhfN), homologous to the isomerase domain of glucosamine-6-phosphate synthase, and a putative cationic amino acid transporter (YhfM). The proteins encoded by YhfQ and YhfN were overexpressed in E. coli, purified, and shown to catalyze the ATP-dependent phosphorylation of fructoselysine to a product identified as fructoselysine 6-phosphate by 31P NMR (YhfQ), and the reversible conversion of fructoselysine 6-phosphate and water to lysine and glucose 6-phosphate (YhfN). The K(m) of the kinase for fructoselysine amounted to 18 microm, and the K(m) of the deglycase for fructoselysine 6-phosphate, to 0.4 mm. A value of 0.15 m was found for the equilibrium constant of the deglycase reaction. The kinase and the deglycase were both induced when E. coli was grown on fructoselysine and then reached activities sufficient to account for the rate of fructoselysine utilization

Identification of glucoselysine-6-phosphate deglycase, an enzyme involved in the metabolism of the fructation product glucoselysine

The metabolism of the glycation product fructose-ϵ-lysine in Escherichia coli involves its ATP-dependent phosphorylation by a specific kinase (FrlD), followed by the conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine by fructoselysine-6-phosphate deglycase (FrlB), which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. As shown in the present work, several bacterial operons comprise: (1) a homologue of fructoselysine-6-phosphate deglycase; (2) a second homologue of the isomerase domain of glucosamine-6-phosphate synthase, more closely related to it; and (3) components of a novel phosphotransferase system, but no FrlD homologue. The FrlB homologue (GfrF) and the closer glucosamine-6-phosphate synthase homologue (GfrE) encoded by an Enterococcus faecium operon were expressed in E. coli and purified. Similar to FrlB, GfrF catalysed the reversible conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine. When incubated with fructose 6-phosphate and elevated concentrations of lysine, GfrE catalysed the formation of a compound identified as 2-ϵ-lysino-2-deoxy-6-phospho-glucose (glucoselysine 6-phosphate) by NMR. GfrE also catalysed the reciprocal conversion, i.e. the formation of fructose 6-phosphate (but not glucose 6-phosphate) from glucoselysine 6-phosphate. The equilibrium constant of this reaction (0.8 M) suggests that the enzyme serves to degrade glucoselysine 6-phosphate. In conclusion, GfrF and GfrE serve to metabolize glycation products formed from lysine and glucose (fructoselysine) or fructose (glucoselysine), via their 6-phospho derivatives. The latter are presumably formed by the putative phosphotransferase system encoded by gfrA–gfrD. The designation gfr (glycation and fructation product degradation) is proposed for this operon. This is the first description of an enzyme participating in the metabolism of fructation products

Enzymatic repair of amadori products

Protein deglycation, a new form of protein repair, involves several enzymes. Fructosamine-3-kinase (FN3K), an enzyme found in mammals and birds, phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins. This enzyme acts particularly well on fructose-epsilon-lysine, both in free form and in the accessible regions of proteins. Mice deficient in FN3K accumulate protein-bound fructosamines and free fructoselysine, indicating that the deglycation mechanism initiated by FN3K is operative in vivo. Mammals and birds also have an enzyme designated 'FN3K-related protein' (FN3KRP), which shares ≈65% sequence identity with FN3K. Unlike FN3K, FN3KRP does not phosphorylate fructosamines, but acts on ribulosamines and erythrulosamines. As with FN3K, the third carbon is phosphorylated and this leads to destabilization of the ketoamines. Experiments with intact erythrocytes indicate that FN3KRP is also a protein-repair enzyme. Its physiological substrates are most likely formed from ribose 5-phosphate and erythrose 4-phosphate, which give rise to ketoamine 5- or 4-phosphates. The latter are dephosphorylated by 'low-molecular-weight protein-tyrosine-phosphatase-A' (LMW-PTP-A) before FN3KRP transfers a phosphate on the third carbon. The specificity of FN3K homologues present in plants and bacteria is similar to that of mammalian FN3KRP, suggesting that deglycation of ribulosamines and/or erythrulosamines is an ancient mechanism. Mammalian cells contain also a phosphatase acting on fructosamine 6-phosphates, which result from the reaction of proteins with glucose 6-phosphate

Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain

A serine synthesis defect presenting with a Charcot-Marie-Tooth-like polyneuropathy.

This case expands the phenotypic spectrum of 3-phosphoglycerate dehydrogenase deficiency. Plasma amino acid chromatography should be added to the list of investigations performed in patients with Charcot-Marie-Tooth–like polyneuropathy, especially if it is associated with psychomotor delay and congenital cataracts

NAT6 acetylates the N‐terminus of different forms of actin

All forms of mammalian actin comprise at their N-terminus a negatively charged region consisting of an N-acetylated aspartate or glutamate followed by two or three acidic residues. This structural feature is unique to actins and important for their interaction with other proteins. The enzyme catalyzing the acetylation of the N-terminal acidic residue is thought to be NAA10, an enzyme that acetylates multiple intracellular proteins. We report here that this acetylation is essentially carried out by NAT6 (Fus2), a protein of unknown function. Tests of the activity of human recombinant NAT6 on a series of purified proteins showed that the best substrate had several acidic residues near its N-terminus. Accordingly NAT6 was particularly active on highly acidic peptides with sequences corresponding to the N-terminus of different forms of mammalian actins. Knocking out of NAT6 in two human cell lines led to absence of acetylation of the first residue of mature beta-actin (Asp2) and gamma-actin-1 (Glu2). Complete acetylation of these two actins was restored by re-expression of NAT6, or by incubation of extracts of NAT6-deficient cells with low concentrations of recombinant NAT6, while NAA10 showed much less or no activity in such assays. Alpha-actin-1 expressed in NAT6-knockout cells was not acetylated at its N-terminus, indicating that the requirement of NAT6 for acetylation of actin N-termini also applies to the skeletal muscle actin isoform. Taken together, our findings reveal that NAT6 plays a critical role in the maturation of actins by carrying out the acetylation of their N-terminal acidic residue

Unusual association between lysinuric protein intolerance and moyamoya vasculopathy

Introduction Lysinuric protein intolerance (LPI) is a form of inherited aminoaciduria caused by a deficiency in the cationic amino acid transport process on the basolateral membrane of enterocytes and renal tubular cells. Clinical signs include gastrointestinal symptoms, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, intellectual deficiency, lung and renal involvement, bone marrow abnormalities, as well as altered immune response. Moyamoya disease is a cerebrovascular disorder predisposing sufferers to stroke through progressive stenosis of the intracranial internal carotid arteries and their proximal branches. Patients with characteristic moyamoya vasculopathy who also exhibit well-recognized associated conditions, such as Down syndrome or sickle-cell disease, are diagnosed with moyamoya syndrome, whereas those with no known associated risk factors are said to suffer from moyamoya disease. Case study A 5-year-old girl exhibiting aversion to protein-rich food and splenomegaly presented with a history of recurrent ischemic strokes. Cerebral angiography confirmed moyamoya vasculopathy. Metabolic investigation revealed abnormalities characteristic of LPI. This diagnosis was confirmed by the detection of a mutation within the SLC7A7 gene upon molecular investigation. Conclusion To the best of our knowledge, this is the first reported case of an association between moyamoya vasculopathy and LPI. While the question of association or coincidence cannot yet be answered, several pathophysiological consequences of LPI can be defined as separate, such as links between the impact of low arginine levels on the function of vascular endothelium and brain nitric oxide metabolism, as well as hemophagocytic syndrome associated with the risk of vasculitis, thus accounting for the development of moyamoya vasculopathy