Giulia Baldini

A Novel Serum Protein Similar to C1q, Produced Exclusively in Adipocytes

We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.

Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis

Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP‐25 (synaptosome‐associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that is common to the other two SNARE proteins: VAMP (vesicle‐associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we analyse the involvement of the four copies of the SNARE motif present in SNAP‐25 in its interaction with BoNT/A and BoNT/E by following the kinetics of proteolysis of SNAP‐25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognise SNAP‐25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. Also, a non‐neuronal isoform of SNAP‐25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP‐25 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases.

Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by ∼40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.

Stimulation-dependent regulation of the pH, volume and quantal size of bovine and rodent secretory vesicles

Trapping of weak bases was utilized to evaluate stimulus‐induced changes in the internal pH of the secretory vesicles of chromaffin cells and enteric neurons. The internal acidity of chromaffin vesicles was increased by the nicotinic agonist 1,1‐dimethyl‐4‐phenyl‐piperazinium iodide (DMPP; in vivo and in vitro) and by high K+ (in vitro); and in enteric nerve terminals by exposure to veratridine or a plasmalemmal [Ca2+]o receptor agonist (Gd3+). Stimulation‐induced acidification of chromaffin vesicles was [Ca2+]o‐dependent and blocked by agents that inhibit the vacuolar proton pump (vH+‐ATPase) or flux through Cl− channels. Stimulation also increased the average volume of chromaffin vesicles and the proportion that displayed a clear halo around their dense cores (called active vesicles). Stimulation‐induced increases in internal acidity and size were greatest in active vesicles. Stimulation of chromaffin cells in the presence of a plasma membrane marker revealed that membrane was internalized in endosomes but not in chromaffin vesicles. The stable expression of botulinum toxin E to prevent exocytosis did not affect the stimulation‐induced acidification of the secretory vesicles of mouse neuroblastoma Neuro2A cells. Stimulation‐induced acidification thus occurs independently of exocytosis. The quantal size of secreted catecholamines, measured by amperometry in cultured chromaffin cells, was found to be increased either by prior exposure to L‐DOPA or stimulation by high K+, and decreased by inhibition of vH+‐ATPase or flux through Cl− channels. These observations are consistent with the hypothesis that the content of releasable small molecules in secretory vesicles is increased when the driving force for their uptake is enhanced, either by increasing the transmembrane concentration or pH gradients.

Reconstitution in vitro of sulfobromophthalein transport by bilitranslocase

Liposomes containing 150 mM KCl and 0.48 mM sulfobromophthalein have been prepared. The internal pH was set at 6.5, a value at which sulfobromopthalein is colorless. When brought to alkaline pH a certain amount of the dye is deprotonated and can be read spectrophotometrically as external sulfobromophthalein. Upon addition of Triton X-100 the membrane is dissolved and all sulfobromophthalein present in the preparation may be measured. Addition of bilitranslocase to such a preparation of liposomes causes the internal sulfobromophthalein to leave the internal compartment. The rate of this phenomenon may be followed directly and shown to be greatly accelerated by the addition of valinomycin. The latter finding indicates that sulfobromophthalein transport occurs in response to a membrane diffusion potential created by permeabilisation to K+ of liposomes brought about by valinomycin (uniport). The permeability change induced by bilitranslocase is specific and does not reflect an alteration of the normal impermeability of liposomes to small ions such as protons or Ca2+.

Rab3A and Rab3D Control the Total Granule Number and the Fraction of Granules Docked at the Plasma Membrane in PC12 Cells

Rab proteins are Ras‐like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immuno‐electron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild‐type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.

Cellular localization of sulfobromophthalein transport activity in rat liver

The movement of sulfobromophthalein is measured in rat liver plasma-membrane vesicles by direct dual-wavelength spectrophotometry. The technique is based on the principle that the dye, when entering a more acidic compartment, changes its absorption in the visible region. From this study it may be concluded that, among the different cellular subfractions, only liver plasma-membrane vesicles can catalyze electrogenic transport of sulfobromophthalein. Plasma membranes from erythrocytes are unable to perform such a function. The movement follows the distribution pattern of (Na+ + K+)-ATPase and it is therefore concluded that this process occurs exclusively at the sinusoidal membrane level. Inhibition studies confirm that the process is catalyzed by bilitranslocase.

Constitutive Traffic of Melanocortin-4 Receptor in Neuro2A Cells and Immortalized Hypothalamic Neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds α-melanocyte-stimulating hormone (α-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of α-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toα-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that α-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.

Role of the Cysteine-rich Domain of the t-SNARE Component, SYNDET, in Membrane Binding and Subcellular Localization

Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys2, Cys4, and Cys5, are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys1 and Cys3, distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys1 and Cys3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution.

Obesity-linked variants of melanocortin-4 receptor are misfolded in the endoplasmic reticulum and can be rescued to the cell surface by a chemical chaperone.

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find that these variants are all retained in the endoplasmic reticulum (ER), are ubiquitinated to a greater extent than the wild-type (wt) receptor, and induce ER stress with increased levels of ER chaperones as compared with wt-MC4R and appearance of CCAAT/enhancer-binding protein homologous protein (CHOP). Expression of the X-box-binding-protein-1 (XBP-1) with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone 4-phenyl butyric acid (PBA) increased the cell surface expression of wt-MC4R, MC4R-I316S, and I317T by more than 40%. PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functions to promote MC4R folding. MC4R-I316S rescued to the cell surface is functional, with a 52% increase in agonist-induced cAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor of the ubiquitin-activating enzyme 1 increased by approximately 40% the expression of the receptors at the cell surface, and the effects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.