Laurent Emorine

MabA (FabG1), a Mycobacterium tuberculosis protein involved in the long-chain fatty acid elongation system FAS-II

The fatty acid elongation system FAS-II is involved in the biosynthesis of mycolic acids, which are very long-chain fatty acids of the cell envelope specific to Mycobacterium tuberculosis and other mycobacteria. A potential component of FAS-II, the protein MabA (FabG1), was overexpressed and purified. Sedimentation equilibrium analyses revealed that MabA undergoes a dimer to tetramer self-association with a dissociation constant of 22 microM. The protein was detected by Western blotting in a mycobacterial cell-wall extract that produces mycolic acids and in the FPLC FAS-II fraction. MabA was shown to catalyse the NADPH-specific reduction of beta-ketoacyl derivatives, equivalent to the second step of a FAS-II elongation round. Unlike the known homologous proteins, MabA preferentially metabolizes long-chain substrates (C(8)-C(20)) and has a poor affinity for the C(4) substrate, in agreement with FAS-II specificities. Molecular modelling of MabA structure suggested the presence of an unusually hydrophobic substrate-binding pocket holding a unique Trp residue, suitable for fluorescence spectroscopic analyses. In agreement with the enzyme kinetic data, the spectral properties of MabA were different in the presence of the C(8)-C(16) ligands as compared to the C(4) ligand. Altogether, these data bring out distinctive enzymic and structural properties of MabA, which correlate with its predilection for long-chain substrates, in contrast to most of the other known ketoacyl reductases.

The human macrophage mannose receptor is not a professional phagocytic receptor

The macrophage mannose receptor (MR) appears to play an important role in the binding and phagocytosis of several human pathogens, but its phagocytic property and signaling pathways have been poorly defined. The general strategy to explore such topics is to express the protein of interest in nonphagocytic cells, but in the case of MR, there are few reports using the full‐length MR cDNA. When we searched to clone de novo the human MR (hMR) cDNA, problems were encountered, and full‐length hMR cDNA was only obtained after devising a complex cloning strategy. Chinese hamster ovary cells, which have a fully functional phagocytic machinery when expressing professional phagocytic receptors, were stably transfected, and cell clones expressing hMR at quantitatively comparable levels than human macrophages or J774E cells were obtained. They exhibited a functional hMR‐mediated endocytic capacity of a soluble ligand but failed to ingest classical particulate ligands of MR such as zymosan, Mycobacterium kansasii, or trimannoside bovine serum albumin‐coated latex beads. Transient expression of hMR in two human cell lines did not provide a phagocytic capacity either. In conclusion, we show that MR is not a professional phagocytic receptor, as it does not possess the ability to promote particle ingestion in nonphagocytic cells on its own. We propose that MR is a binding receptor, which requires a partner to trigger phagocytosis in some specialized cells such as macrophages. Our new expression vector could represent a useful tool to study the receptor and its partnership further.

Lack of Palmitoylation Redirects p59Hck from the Plasma Membrane to p61Hck-positive Lysosomes

Hck, a protein-tyrosine kinase of phagocytes, is the unique member of the Src family expressed under two alternatively translated isoforms differing in their N-terminal site of acylation: p61Hck has an additional 21-amino acid sequence comprising a single myristoylation motif, whereas p59Hck N terminus has myristoylation and palmitoylation sites. To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61Hck was associated with lysosomal vesicles, whereas p59Hck was found at the plasma membrane and to a low extent associated with lysosomes. Their unique N-terminal domains were sufficient to target GFP to the corresponding intracellular compartments. Mutation of the palmitoylation site of p59Hckredirected this isoform to lysosomes, indicating that the palmitoylation state governs the association of p59Hck with the plasma membrane or with lysosomes. In addition, both isoforms and the nonpalmitoylated p59Hck mutant were found on the Golgi apparatus, suggesting a role of this organelle in the subcellular sorting of Hck isoforms. Regarding their subcellular localizations, we propose that bi-acylated p59Hck might transduce plasma membrane receptor signals, whereas p61Hck and the nonpalmitoylated p59Hck might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes.

Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris

The human μ‐opioid receptor cDNA from which the 32 amino‐terminal codons were substituted by the Saccharomyces cerevisiae α‐mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase‐1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (K d = 0.2 nM and B max = 400 fmol/mg protein or 800 sites/cell). Competition studies with non‐selective, and μ‐, δ‐ and κ‐selective opioid agonists and antagonists revealed a typical μ‐opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

Agonist‐induced signaling and trafficking of the μ‐opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C‐terminal domain

The human μ‐opioid receptor and a mutant form, μS/T[i3+Cter]A, in which all Ser and Thr residues from the third cytoplasmic loop and C‐terminal domain were changed to Ala, were studied after expression in CHO‐K1 cells. Although the mutant receptors had similar affinities for agonists and EC50 values for inhibition of adenylyl cyclase as compared to wild‐type receptors, the E max were almost 2‐fold decreased, suggesting a role of the mutated residues in G‐protein coupling. After chronic morphine or etorphine, the EC50 values of the agonists were about 5‐fold increased at both receptors but the E max values were not altered; upon agonist withdrawal forskolin‐stimulated cAMP levels were increased to almost 200% of control levels. Sequestration and rapid down‐regulation of the μ‐opioid receptor were induced by DAGO and etorphine but not morphine. In contrast, the μS/T[i3+Cter]A receptor was not sequestered and was up‐regulated (150–380%) after treatment with agonists. The results indicate that the Ser and Thr residues in the third cytoplasmic loop and C‐terminus of the μ‐opioid receptor are not involved in the limited desensitization or in the adenylyl cyclase superactivation promoted by agonists but that their integrity and/or their phosphorylation is required in the intricate and coordinately regulated pathways involved in receptor signaling and trafficking.

p59Hck isoform induces F-actin reorganization to form protrusions of the plasma membrane in a Cdc42- and Rac-dependent manner.

Hck is a protein kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, localized at the plasma membrane and lysosomes, respectively. Their individual involvement in functions ascribed to Hck, phagocytosis, cell migration, and lysosome mobilization, is still unclarified. To investigate the specific role of p59Hck, a constitutively active variant in fusion with green fluorescent protein (p59Hckca) was expressed in HeLa cells. p59Hckca was found at focal adhesion sites and triggered reorganization of the actin cytoskeleton, leading to plasma membrane protrusions where it co-localized with F-actin. Similarly, microinjection of p59Hckca cDNA in J774.A1 macrophages induced membrane protrusions. Whereas kinase activity and membrane association of p59Hck were dispensable for location at focal adhesions, p59Hck-induced membrane protrusions were dependent on kinase activity, plasma membrane association, and Src homology 2 but not Src homology 3 domain and were inhibited by dominant-negative forms of Cdc42 or Rac but not by blocking Rho activity. A dominant negative form of p59Hck inhibited the Cdc42- and Rac-dependent FcγRIIa-mediated phagocytosis. Expression of the Cdc42/Rac-interacting domain of p21-activated kinase in macrophages abolished the p59Hckca-induced morphological changes. Therefore, p59Hck-triggered remodeling of the actin cytoskeleton depends upon the activity of Cdc42 and Rac to promote formation of membrane protrusions necessary for phagocytosis and cell migration.

Identification in the μ-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents

Inactivation by thiol reducing and alkylating agents of ligand binding to the human μ‐opioid receptor was examined. Dithiothreitol reduced the number of [3H]diprenorphine binding sites. Replacement by seryl residues of either C142 or C219 in extracellular loops 1 and 2 of the μ receptor resulted in a complete loss of opioid binding. A disulfide bound linking C142 to C219 may thus be essential to maintain a functional conformation of the receptor. We also demonstrated that inactivation of ligand binding upon alkylation by N‐ethylmaleimide occurred at two sites. Alteration of the more sensitive (IC50=20 μM) did not modify antagonists binding but decreased agonist affinity almost 10‐fold. Modification of the less reactive site (IC50=2 mM) decreased the number of both agonist and antagonist binding sites. The alkylation site of higher sensitivity to N‐ethylmaleimide was shown by mutagenesis experiments to be constituted of both C81 and C332 in transmembrane domains 1 and 7 of the μ‐opioid receptor.

Implication of the First and Third Extracellular Loops of the μ-Opioid Receptor in the Formation of the Ligand Binding Site: A Study Using Chimeric μ-Opioid/Angiotensin Receptors

Abstract: Recent studies on chimeric μ/δ‐, μ/κ‐ and δ/κ‐opioid receptors have suggested that extracellular loops of the receptors were involved in the discriminatory binding of selective ligands by controlling their entry into the transmembrane binding site. Since homochimeric opioid receptors are mostly informative in terms of selectivity, the role of extracellular loops was examined here by studying heterochimeric μ receptors where the totality or parts of extracellular loops were replaced by the corresponding regions of the receptor for angiotensin II. Chimeric μ receptors with extracellular loop EL1 or EL3 originating from the angiotensin receptor had 100‐fold decreased affinities for opioids; the length of the first extracellular loop, which is one residue longer in angiotensin than μ receptors, was shown to be responsible for this situation. Substitution of the μ receptor second extracellular loop by that of the angiotensin receptor diminished by ∼10‐fold the affinities for opioids. Since all chimeras had altered affinities for selective and nonselective ligands, we propose that extracellular domains of the μ receptor, particularly the first and third loops, constrain the relative positioning of the connected transmembrane domains where selective as well as nonselective contact points form the opioid binding site.