N-acetylglucosaminyltransferase I (GnT I) serves as the gateway from oligomannose to hybrid and complex N-glycans and plays a critical role in mammalian development and possibly all metazoans. We have determined the X-ray crystal structure of the catalytic fragment of GnT I in the absence and presence of bound UDP-GlcNAc/Mn 2+ at 1.5 and 1.8 A Ê resolution, respectively. The structures identify residues critical for substrate binding and catalysis and provide evidence for similarity, at the mechanistic level, to the deglycosylation step of retaining b-glycosidases. The structuring of a 13 residue loop, resulting from UDPGlcNAc/Mn 2+ binding, provides an explanation for the ordered sequential`Bi Bi' kinetics shown by GnT I. Analysis reveals a domain shared with Bacillus subtilis glycosyltransferase SpsA, bovine b-1,4-galactosyltransferase 1 and Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase. The low sequence identity, conserved fold and related functional features shown by this domain de®ne a superfamily whose members probably share a common ancestor. Sequence analysis and protein threading show that the domain is represented in proteins from several glycosyltransferase families.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) coactivator 1␣ (PGC-1␣) is a transcriptional coactivator that is a key component in the regulation of energy production and utilization in metabolic tissues. Recent work has identified PGC-1␣ as a strong coactivator of the orphan nuclear receptor estrogen-related receptor ␣ (ERR␣), implicating ERR␣ as a potential mediator of PGC-1␣ action. To understand the role of ERR␣ in PGC-1␣ signaling, a parallel approach of high-throughput screening and gene-expression analysis was used to identify ERR␣ small-molecule regulators and target genes. We report here the identification of a potent and selective ERR␣ inverse agonist that interferes effectively with PGC-1␣͞ERR␣-dependent signaling. This inverse agonist inhibits the constitutive activity of ERR␣ in both biochemical and cell-based assays. Also, we demonstrate that monoamine oxidase B is an ERR␣ target gene whose expression is regulated by PGC-1␣ and ERR␣ and inhibited by the ERR␣ inverse agonist. The discovery of potent and selective ERR␣ modulators and their effect on PGC-1␣ signaling provides mechanistic insight into gene regulation by PGC-1␣. These findings validate ERR␣ as a promising therapeutic target in the treatment of metabolic disorders, including diabetes and obesity.

The estrogen-related receptor alpha (ERRalpha) is an orphan receptor belonging to the nuclear receptor superfamily. The physiological role of ERRalpha has yet to be established primarily because of lack of a natural ligand. Herein, we describe the discovery of the first potent and selective inverse agonist of ERRalpha. Through in vitro and in vivo studies, these ligands will elucidate the endocrine signaling pathways mediated by ERRalpha including association with human disease states.

The purine anti-metabolite 6-mercaptopurine is one of the most widely used drugs for the treatment of acute childhood leukemia and chronic myelocytic leukemia. Developed in the 1950s, the drug is also being used as a treatment for inflammatory diseases such as Crohn's disease. The antiproliferative mechanism of action of this drug and other purine anti-metabolites has been demonstrated to be through inhibition of de novo purine synthesis and incorporation into nucleic acids. Despite the extensive clinical use and study of 6-mercaptopurine and other purine analogues, the cellular effects of these compounds remain relatively unknown. More recently, purine anti-metabolites have been shown to function as protein kinase inhibitors and to regulate gene expression. In an attempt to find small molecule regulators of the orphan nuclear receptor Nurr1, interestingly, we identified 6-mercaptopurine as a specific activator of this receptor. A detailed analysis of 6-mercaptopurine regulation of Nurr1 demonstrates that 6-mercaptopurine regulates Nurr1 through a region in the amino terminus. This activity can be inhibited by components of the purine biosynthesis pathway. These findings indicate that Nurr1 may play a role in mediating some of the antiproliferative effects of 6-mercaptopurine and potentially implicate Nurr1 as a molecular target for treatment of leukemias.The Nobel prize-winning work of Elion et al.(1) demonstrated that differences in nucleic acid metabolism between cancerous cells and normal cells or between cells from different organisms led to the design and development of nucleic acid analogs that would effectively and selectively block nucleic acid synthesis in the desired target cells. Among the drugs that emanated from this work are 6-mercaptopurine (6-MP), 1 6-thioguanine (6-TG), azathioprine, allopurinol, and acyclovir (2, 3). These drugs are still in use for the treatment of leukemias (6-MP and 6-TG) and autoimmune disorders and the prevention of organ transplant rejection (azathioprine), gout (allopurinol), and herpes virus infections (acyclovir). Additional nucleic acid anti-metabolites that were developed are effective in bacterial infections and malaria.The clinical efficacy of 6-MP is due in part to antiproliferative and cytotoxic effects resulting primarily from the inhibition of purine biosynthesis at multiple steps and incorporation into nucleic acids as thioguanine nucleotides (2-7). More recent work has expanded the function of purine anti-metabolites by demonstrating that compounds such as 6-thioguanine or 6-mercaptopurine can target biological activities outside of the purine biosynthesis pathway including telomerase (5), protein kinase N (6, 7), axon growth and regeneration (8, 9), and apoptosis in B cells through the regulation of the Bcl-2/Bax ratio (10).In this study, we have identified, from a high throughput screen, 6-mercaptopurine as a regulator of the transcriptional activity of the orphan nuclear hormone receptor Nurr1. There are three members of the NGFI-B group, including Nurr1 ...

UDP-N-acetylglucosamine:␣-3-D-mannoside ␤-1,2-Nacetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of Asn-linked complex and hybrid glycans. Studies on mice with a null mutation in the GnT I gene have indicated that N-glycans play critical roles in mammalian morphogenesis. This paper presents studies on N-glycans during the development of the nematode Caenorhabditis elegans. We have cloned cDNAs for three predicted C. elegans genes homologous to mammalian GnT I (designated gly-12, gly-13, and gly-14). All three cDNAs encode proteins (467, 449, and 437 amino acids, respectively) with the domain structure typical of previously cloned Golgi-type glycosyltransferases. Expression in both insect cells and transgenic worms showed that gly-12 and gly-14, but not gly-13, encode active GnT I. All three genes were expressed throughout worm development (embryo, larval stages L1-L4, and adult worms). The gly-12 and gly-13 promoters were expressed from embryogenesis to adulthood in many tissues. The gly-14 promoter was expressed only in gut cells from L1 to adult developmental stages. Transgenic worms that overexpress any one of the three genes show no obvious phenotypic defects. The data indicate that C. elegans is a suitable model for further study of the role of complex N-glycans in development.