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.

Leukocyte type core 2 ␤1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Gal␤1-3[GlcNAc␤1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Gal␤1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Gal␤1-3GalNAc at 2.0 and 2.7 Å resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Gal␤1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside disphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.3 is a cis-medial Golgi resident glycosyltransferase that catalyzes the conversion of the core 1 O-glycan to that of the core 2 structure (1, 2). Core 2 O-glycans have been shown to be key ligands in selectin-mediated lymphocyte homing and leukocyte rolling; lymphocyte L-selectins bind to endothelial cell O-glycans containing 6-sulfo sialyl Lewis x on core 2 and extended core 1 structures, whereas neutrophils expressing sialyl Lewis x on core 2 O-glycans bind to E-and P-selectins on endothelial cells (3-5). C2GnT-L is also of considerable interest in the study of tumor metastasis given that its expression is highly correlated with tumor progression in a number of cancers. It is overexpressed in colorectal, lung, and prostate cancer, and recent work has shown that transfection of C2GnT-L into a prostate cancer cell line leads to increased tumor size in an experimental tumor model (6 -8).C2GnT-L is an inverting N-acetylglucosaminyltransferase belonging to family GT-14 (9). It transfers N-acetylglucosamine (GlcNAc), in ␤1,6 linkage, from UDP-GlcNAc to Gal␤1-3Gal-NAc-O-Ser/Thr (core 1) to generate Gal␤1-3[GlcNAc␤1-6]GalNAc-O-Ser/Thr (core 2). C2GnT-L (leukocyte-type or C2GnT-I) along with C2GnT-M (mucin-type or C2GnT-II) (10, 11) and C2GnT-T (thymus-type or C2GnT-III) (12) are the three ␤1,6 N-acetylglucosaminyltransferases involved in the biosynthesis of core 2 branched O-glycans. The three enzymes differ in tissue distribution, and the mammalian homologues show 40 -50% sequence similarity with each other. Divalent metal ions, which have been shown to be essential for catalytic activity in many glycosyltransf...

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species.Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7–66.4% and 56.5–60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.