Structure of the molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism

The molybdenum cofactor is part of the active site of all molybdenum-dependent enzymes, except nitrogenase. The molybdenum cofactor consists of molybdopterin, a phosphorylated pyranopterin, with an ene-dithiolate coordinating molybdenum. The same pyranopterin-based cofactor is involved in metal coordination of the homologous tungsten-containing enzymes found in archea. The molybdenum cofactor is synthesized by a highly conserved biosynthetic pathway. In plants, the multidomain protein Cnx1 catalyses the insertion of molybdenum into molybdopterin. The Cnx1 G domain (Cnx1G), whose crystal structure has been determined in its apo form, binds molybdopterin with high affinity and participates in the catalysis of molybdenum insertion. Here we present two high-resolution crystal structures of Cnx1G in complex with molybdopterin and with adenylated molybdopterin (molybdopterin-AMP), a mechanistically important intermediate. Molybdopterin-AMP is the reaction product of Cnx1G and is subsequently processed in a magnesium-dependent reaction by the amino-terminal E domain of Cnx1 to yield active molybdenum cofactor. The unexpected identification of copper bound to the molybdopterin dithiolate sulphurs in both structures, coupled with the observed copper inhibition of Cnx1G activity, provides a molecular link between molybdenum and copper metabolism.     

Chemical remodelling of cell surfaces in living animals

Cell surfaces are endowed with biological functionality designed to mediate extracellular communication. The cell-surface repertoire can be expanded to include abiotic functionality through the biosynthetic introduction of unnatural sugars into cellular glycans, a process termed metabolic oligosaccharide engineering. This technique has been exploited in fundamental studies of glycan-dependent cell-cell and virus-cell interactions and also provides an avenue for the chemical remodelling of living cells. Unique chemical functional groups can be delivered to cell-surface glycans by metabolism of the corresponding unnatural precursor sugars. These functional groups can then undergo covalent reaction with exogenous agents bearing complementary functionality. The exquisite chemical selectivity required of this process is supplied by the Staudinger ligation of azides and phosphines, a reaction that has been performed on cultured cells without detriment to their physiology. Here we demonstrate that the Staudinger ligation can be executed in living animals, enabling the chemical modification of cells within their native environment. The ability to tag cell-surface glycans in vivo may enable therapeutic targeting and non-invasive imaging of changes in glycosylation during disease progression.     

Identification of swelling-activated Cl<Superscript>-</Superscript> current in rabbit cardiac Purkinje cells

The presence and functional role of the swelling-activated Cl^- current (I_Cl(swell)) in rabbit cardiac Purkinje cells was examined using patch-clamp methodology. Extracellular hypotonicity (210 or 135 mOsm) activated an outwardly rectifying, time-independent current with a reversal potential close to the calculated Cl^- equilibrium potential (E_Cl). The magnitude of this current was related to tonicity of the superfusate. The current was blocked by 0.5 mM 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). These features are comparable to those of I_Cl(swell) found in sinoatrial nodal, atrial, and ventricular myocytes. I_Cl(swell) activation at 210 and 135 mOsm depolarized the resting membrane potential with 6 and 10 mV and shortened the action potential by 18 and 33%, respectively. DIDS partially reversed I_Cl(swell)-induced action potential changes. We conclude that I_Cl(swell) is present in Purkinje cells and its activation leads to action potential shortening and resting membrane potential depolarization, both of which can promote the development of reentrant arrhythmias.Received 20 January 2004; received after revision 17 February 2004; accepted 25 February 2004     

FK506 sensitizes mammalian cells to high osmolarity by modulating p38 MAP kinase activation

The immunosuppressants tacrolimus (FK506) and cyclosporin A (CsA) have increased the survival rates in organ transplantation. Both drugs inhibit the protein phosphatase calcineurin (CaN) in activated T cells, exhibiting similar side-effects. Diabetes is observed more often in FK506 than CsA therapy, probably due to inhibition of new molecular targets other than CaN. We studied FK506 toxicity in mammalian cells. FK506, but not CsA, regulated p38 activation by osmotic stress, and decreased viability in osmostressed cells. In addition, FK506 treatment strongly increased the phosphorylation of the eukaryotic initiation factor-2a (eIF-2a) subunit. eIF-2a phosphorylation, p38 inhibition and cell lethality were relieved by addition of excess amino acids to the medium, suggesting that amino acid availability mediated FK506 toxicity. Therefore, these FK506-dependent responses could be relevant to the non-therapeutic effects of FK506 therapy.Received 16 October 2003; received after revision 8 January 2004; accepted 14 January 2004     

Neurobiology and neuroimmunology of Tourette’s syndrome: an update

Tourettes syndrome is a childhood-onset neuropsychiatric disorder characterized by the presence of both multiple motor and vocal tics. While the pathogenesis at a molecular and cellular level remains unknown, structural and functional neuroimaging studies point to the involvement of the basal ganglia and related cortico-striato-thalamo-cortical circuits as the neuroanatomical site for Tourettes syndrome. Moreover, Tourettes syndrome has a strong genetic component, and considerable progress has been made in understanding the mode of transmission and in identifying potential genomic loci. Summaries of recent findings in these areas will be reviewed, followed by a critical overview of findings both supporting and challenging the proposed autoimmune hypothesis of Tourettes syndrome. We conclude that Tourettes syndrome is a heterogeneous disorder, and that immune factors may indeed be involved in some patients.Received 12 August 2003; received after revision 8 October 2003; accepted 31 October 2003     

Role of <Emphasis Type="Italic">N</Emphasis>-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation

Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc₃Man₉GlcNAc₂ glycans after trimming with endoplasmic reticulum (ER) -glucosidases and -mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc₁Man₉GlcNAc₂) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man₈GlcNAc₂ isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc₂ along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man₅GlcNAc₁ by the action of cytosolic endo--N-acetylglucosaminidase and -mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc₃Man₉GlcNAc₂-P-P-Dol, and the unfolded protein response, which enhances synthesis of components of the quality control system.Received 26 January 2004; accepted 25 February 2004     

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of α-lactalbumin

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human -lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.Received 30 January 2004; received after revision 5 March 2004; accepted 16 March 2004     

The staphylocoagulase family of zymogen activator and adhesion proteins

Staphylocoagulase (SC) secreted by Staphylococcus aureus is a potent non-proteolytic activator of the blood coagulation zymogen prothrombin and the prototype of a newly established zymogen activator and adhesion protein (ZAAP) family. The conformationally activated SC·prothrombin complex specifically cleaves fibrinogen to fibrin, which propagates the growth of bacteria-fibrin-platelet vegetations in acute bacterial endocarditis. Our recent 2.2 Å X-ray crystal structures of an active SC fragment [SC(1-325)] bound to the prothrombin zymogen catalytic domain, prethrombin 2, demonstrated that SC(1-325) represents a new type of non-proteolytic activator with a unique fold. The observed insertion of the SC(1-325) N-terminus into the Ile 16 cleft of prethrombin 2, which triggers the activating conformational change, provided the first unambiguous structural evidence for the molecular sexuality mechanism of non-proteolytic zymogen activation. Based on the SC(1-325) fold, a new family of bifunctional zymogen activator and adhesion proteins was identified that possess N-terminal domains homologous to SC(1-325) and C-terminal domains that mediate adhesion to plasma or extracellular matrix proteins. Further investigation of the ZAAP family may lead to new insights into the mechanisms of bacterial factors that hijack zymogens of the human blood coagulation and fibrinolytic systems to promote and disseminate endocarditis and other infectious diseases.Received 30 June 2004; received after revision 28 July 2004; accepted 4 August 2004