Recombinant adiponectin does not lower plasma glucose in animal models of type 2 diabetes.

AIMS/HYPOTHESIS: Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats. METHODS: Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10-30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5-15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days. RESULTS: Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active. CONCLUSIONS/INTERPRETATION: Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.

Adiponectin promotes macrophage polarization toward an anti-inflammatory phenotype.

It is established that the adipocyte-derived cytokine adiponectin protects against cardiovascular and metabolic diseases, but the effect of this adipokine on macrophage polarization, an important mediator of disease progression, has never been assessed. We hypothesized that adiponectin modulates macrophage polarization from that resembling a classically activated M1 phenotype to that resembling alternatively-activated M2 cells. Peritoneal macrophages and the stromal vascular fraction (SVF) cells of adipose tissue isolated from adiponectin knock-out mice displayed increased M1 markers, including tumor necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein-1 and decreased M2 markers, including arginase-1, macrophage galactose N-acetyl-galactosamine specific lectin-1, and interleukin-10. The systemic delivery of adenovirus expressing adiponectin significantly augmented arginase-1 expression in peritoneal macrophages and SVF cells in both wild-type and adiponectin knock-out mice. In culture, the treatment of macrophages with recombinant adiponectin protein led to an increase in the levels of M2 markers and a reduction of reactive oxygen species and reactive oxygen species-related gene expression. Adiponectin also stimulated the expression of M2 markers and attenuated the expression of M1 markers in human monocyte-derived macrophages and SVF cells isolated from human adipose tissue. These data show that adiponectin functions as a regulator of macrophage polarization, and they indicate that conditions of high adiponectin expression may deter metabolic and cardiovascular disease progression by favoring an anti-inflammatory phenotype in macrophages.

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake.

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

A novel strategy for the purification of recombinantly expressed unstructured protein domains.

We recently found that the larger parts of the endocytic proteins epsin 1 and AP180 consist of an unstructured polypeptide chain. As a result these segments are completely heat-stable without loss of their functional properties. We have taken advantage of this fact and developed a combined heat lysis and pre-purification procedure after expressing the disordered domains in E. coli. This results in the irreversible denaturation and precipitation of the majority of bacterial proteins. The bacteria are resuspended in a non-denaturing buffer, heated in a boiling water bath and shock-cooled. We demonstrate that this procedure compared to conventional lysis improves both yield and quality of the purified protein.

Clint: a novel clathrin-binding ENTH-domain protein at the Golgi.

We have characterized a novel clathrin-binding 68-kDa epsin N-terminal homology domain (ENTH-domain) protein that we name clathrin interacting protein localized in the trans-Golgi region (Clint). It localizes predominantly to the Golgi region of epithelial cells as well as to more peripheral vesicular structures. Clint colocalizes with AP-1 and clathrin only in the perinuclear area. Recombinantly expressed Clint interacts directly with the gamma-appendage domain of AP-1, with the clathrin N-terminal domain through the peptide motif (423)LFDLM, with the gamma-adaptin ear homology domain of Golgi-localizing, gamma-adaptin ear homology domain 2, with the appendage domain of beta2-adaptin and to a lesser extent with the appendage domain of alpha-adaptin. Moreover, the Clint ENTH-domain asssociates with phosphoinositide-containing liposomes. A significant amount of Clint copurifies with rat liver clathrin-coated vesicles. In rat kidney it is preferentially expressed in the apical region of epithelial cells that line the collecting duct. Clathrin and Clint also colocalize in the apical region of enterocytes along the villi of the small intestine. Apart from the ENTH-domain Clint has no similarities with the epsins AP180/CALM or Hip1/1R. A notable feature of Clint is a carboxyl-terminal methionine-rich domain (Met(427)-Met(605)), which contains >17% methionine. Our results suggest that Clint might participate in the formation of clathrin-coated vesicles at the level of the trans-Golgi network and remains associated with the vesicles longer than clathrin and adaptors.

Unusual structural organization of the endocytic proteins AP180 and epsin 1.

Epsin and AP180/CALM are important endocytic accessory proteins that are believed to be involved in the formation of clathrin coats. Both proteins associate with phosphorylated membrane inositol lipids through their epsin N-terminal homology domains and with other components of the endocytic machinery through short peptide motifs in their carboxyl-terminal segments. Using hydrodynamic and spectroscopic methods, we demonstrate that the parts of epsin 1 and AP180 that are involved in protein-protein interactions behave as poorly structured flexible polypeptide chains with little or no conventional secondary structure. The predominant cytosolic forms of both proteins are monomers. Furthermore, we show that recombinant epsin 1, like AP180, drives in vitro assembly of clathrin cages. We conclude that the epsin N-terminal homology domain-containing proteins AP180/CALM and epsin 1 have a very similar molecular architecture that is designed for the rapid and efficient recruitment of the principal coat components clathrin and AP-2 at the sites of coated pit assembly.