Summary Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multi-finger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally-integrated EGFP reporter gene. In summary, OPEN provides an “open-source” method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.
(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B 6 . These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.
Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.
The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N 6 -formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3 -formylphosphate residues arising from 5 -oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N 6 -amino groups of lysine side chains. A liquid chromatography (LC)-tandem mass spectrometry (MS) method was developed to quantify the resulting N 6 -formyl-lysine residues, which were observed to be present histone acetylation ͉ oxidative stress ͉ enediyne
In type 2 diabetes, the liver produces excessive amounts of glucose through the gluconeogenesis (GNG) pathway and consequently is partly responsible for the elevated glucose levels characteristic of the disease. In an effort to find safe and efficacious GNG inhibitors, we targeted the AMP binding site of fructose 1,6-bisphosphatase (FBPase). The hydrophilic nature of AMP binding sites and their widespread use for allosteric regulation of enzymes in metabolic pathways has historically made discovery of AMP mimetics suitable for drug development difficult. By using a structure-based drug design strategy, we discovered a series of compounds that mimic AMP but bear little structural resemblance. The lead compound, MB05032, exhibited high potency and specificity for human FBPase. Oral delivery of MB05032 was achieved by using the bisamidate prodrug MB06322 (CS-917), which is converted to MB05032 in two steps through the action of an esterase and a phosphoramidase. MB06322 inhibited glucose production from a variety of GNG substrates in rat hepatocytes and from bicarbonate in male Zucker diabetic fatty rats. Analysis of liver GNG pathway intermediates confirmed FBPase as the site of action. Oral administration of MB06322 to Zucker diabetic fatty rats led to a dose-dependent decrease in plasma glucose levels independent of insulin levels and nutritional status. Glucose lowering occurred without signs of hypoglycemia or significant elevations in plasma lactate or triglyceride levels. The findings suggest that potent and specific FBPase inhibitors represent a drug class with potential to treat type 2 diabetes through inhibition of GNG.endogenous glucose production ͉ AMP mimetic ͉ structure-based drug design ͉ phosphonate prodrug ͉ antihyperglycemic T he liver (1, 2) and, to a lesser extent, the kidneys (3) are the primary organs responsible for endogenous glucose production (EGP). In type 2 diabetes mellitus (T2DM), excessive EGP in the fasted state and fed state contributes to the chronic elevation of blood glucose levels found in patients with advanced (4, 5) and mild diabetes (6, 7). Moreover, fasting plasma glucose levels correlate with EGP rates in patients with fasting plasma glucose Ͼ180 mg͞dl (10 mM) (8) and possibly in patients with lower levels (6, 7). Studies using 13 C NMR spectroscopy (9) as well as more recent studies using deuterated water (7, 10) attribute the excessive EGP in T2DM patients to increased flux through the gluconeogenesis (GNG) pathway.Efforts over the past 40 years to discover inhibitors of GNG produced few safe and effective drug candidates (11). Metformin, the only marketed drug that acts, at least partially, through inhibition of GNG (12), inhibits GNG indirectly and only 33-36% at the rarely prescribed maximal human dose (13). Direct GNG inhibitors show more pronounced glucose lowering in animals but not without eliciting safety-related concerns. Hypoglycemia, lactic acidosis, and hypertriglyceridemia are the principle safety risks associated with GNG inhibition as highlighted in studie...
Neurotrophins (NTs) are important regulators for the survival, differentiation and maintenance of different peripheral and central neurons. NTs bind to two distinct classes of glycosylated receptor: the p75 neurotrophin receptor (p75(NTR)) and tyrosine kinase receptors (Trks). Whereas p75(NTR) binds to all NTs, the Trk subtypes are specific for each NT. The question of whether NTs stimulate p75(NTR) by inducing receptor homodimerization is still under debate. Here we report the 2.6-A resolution crystal structure of neurotrophin-3 (NT-3) complexed to the ectodomain of glycosylated p75(NTR). In contrast to the previously reported asymmetric complex structure, which contains a dimer of nerve growth factor (NGF) bound to a single ectodomain of deglycosylated p75(NTR) (ref. 3), we show that NT-3 forms a central homodimer around which two glycosylated p75(NTR) molecules bind symmetrically. Symmetrical binding occurs along the NT-3 interfaces, resulting in a 2:2 ligand-receptor cluster. A comparison of the symmetrical and asymmetric structures reveals significant differences in ligand-receptor interactions and p75(NTR) conformations. Biochemical experiments indicate that both NT-3 and NGF bind to p75(NTR) with 2:2 stoichiometry in solution, whereas the 2:1 complexes are the result of artificial deglycosylation. We therefore propose that the symmetrical 2:2 complex reflects a native state of p75(NTR) activation at the cell surface. These results provide a model for NTs-p75(NTR) recognition and signal generation, as well as insights into coordination between p75(NTR) and Trks.
Cellular DNA lesions are efficiently countered by DNA repair in conjunction with delays in cell cycle progression. Previous studies have demonstrated that Rad9, Hus1, and Rad1 can form a heterotrimeric complex (the 9-1-1 complex) that plays dual roles in cell cycle checkpoint activation and DNA repair in eukaryotic cells. Although the 9-1-1 complex has been proposed to form a toroidal structure similar to proliferating cell nuclear antigen (PCNA), which plays essential roles in DNA replication and repair, the structural basis by which it performs different functions has not been elucidated. Here we report the crystal structure of the human 9-1-1 complex at 3.2 Å resolution. The crystal structure, together with biochemical assays, reveals that the interdomain connecting loops (IDC loop) of hRad9, hHus1, and hRad1 are largely divergent, and further cocrystallization study indicates that a PCNA-interacting box (PIP box)-containing peptide derived from hFen1 binds tightly to the interdomain connecting loop of hRad1, providing the molecular basis for the damage repair-specific activity of the 9-1-1 complex in contrast to PCNA. Furthermore, structural comparison with PCNA reveals other unique structural features of the 9-1-1 complex that are proposed to contribute to DNA damage recognition.Cellular DNA damage triggers the activation of the cell cycle checkpoint, leading to a delay or arrest in cell cycle progression to prevent replication and inducing DNA damage repair (1, 2). In response to DNA damage, the 9-1-1 3 complex can be loaded onto DNA lesion sites by Rad17-RFC2-5 (which consists of one large subunit, Rad17, and four small subunits, RFC2-5), where it triggers the activation of the cell cycle checkpoint (3, 4). Moreover, the 9-1-1 complex can also directly participate in DNA repair via physical association with many factors involved in base excision repair (BER), translesion synthesis, homologous recombination, and mismatch repair pathways (5-9). Although both the 9-1-1 and the PCNA complexes perform critical functions in eukaryotic cells with predicted similar structures (10), their specific roles are distinct. First, the 9-1-1 complex is a DNA damage sensor in the cell cycle checkpoint but does not function as a scaffold for the major DNA replication factors; however, PCNA plays exactly the opposite role (1, 11). Second, although both the complexes function in DNA repair, their specific activities are different. Previous observations indicated that some BER enzymes, such as MYH (MutY glycosylate homolog) (12), TDG (thymine DNA glycosylate) (7), and NEIL (Nei-like glycosylate) (8), interact with the 9-1-1 complex via motifs that are located outside the conserved PCNA-interacting box (the PIP box), implying that the 9-1-1 complex functions as a damage repair-specific clamp, in contrast to PCNA. However, the structural basis for this hypothesis remains unclear. Another important unresolved issue concerns the damage-sensing mechanism of the 9-1-1 complex. During the DNA replication process, the PCNA⅐RFC clamp⅐c...
The early Drosophila embryo is a large syncytial cell that compartmentalizes mitotic spindles with furrows. Before furrow ingression, an Arp2/3 actin cap forms above each nucleus and is encircled by actomyosin. We investigated how these networks transform a flat cortex into a honeycomb-like compartmental array. The growing caps circularize and ingress upon meeting their actomyosin borders, which become the furrow base. Genetic perturbations indicate that the caps physically displace their borders and, reciprocally, that the borders resist and circularize their caps. These interactions create an actomyosin cortex arrayed with circular caps. The Rac-GEF Sponge, Rac-GTP, Arp3, and actin coat the caps as a growing material that can drive cortical bending for initial furrow ingression. Additionally, laser ablations indicate that actomyosin contraction squeezes the cytoplasm, producing counterforces that swell the caps. Thus, Arp2/3 caps form clearances of the actomyosin cortex and control buckling and swelling of these clearances for metaphase compartmentalization.
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