A phosphorylation-regulated brake mechanism controls the initial endocytosis of opioid receptors but is not required for post-endocytic sorting to lysosomes.

The delta-opioid receptor (DOR) can undergo proteolytic down-regulation by endocytosis of receptors followed by sorting of internalized receptors to lysosomes. Although phosphorylation of the receptor is thought to play an important role in controlling receptor down-regulation, previous studies disagree on whether phosphorylation is actually required for the agonist-induced endocytosis of opioid receptors. Furthermore, no previous studies have determined whether phosphorylation is required for subsequent sorting of internalized receptors to lysosomes. We have addressed these questions by examining the endocytic trafficking of a series of mutant versions of DOR expressed in stably transfected HEK 293 cells. Our results confirm that phosphorylation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxyl-terminal cytoplasmic domain containing sites of regulatory phosphorylation. However, phosphorylation is required for endocytosis of full-length receptors. Mutation of all serine/threonine residues located in the distal carboxyl-terminal tail domain of the full-length receptor to alanine creates functional mutant receptors that exhibit no detectable agonist-induced endocytosis. Substitution of these residues with aspartate restores the ability of mutant receptors to undergo agonist-induced endocytosis. Studies using green fluorescent protein-tagged versions of arrestin-3 suggest that the distal tail domain, when not phosphorylated, inhibits receptor-mediated recruitment of beta-arrestins to the plasma membrane. Biochemical and radioligand binding studies indicate that, after endocytosis occurs, phosphorylation-defective mutant receptors traffic to lysosomes with similar kinetics as wild type receptors. We conclude that phosphorylation controls endocytic trafficking of opioid receptors primarily by regulating a "brake" mechanism that prevents endocytosis of full-length receptors in the absence of phosphorylation. After endocytosis occurs, subsequent steps of membrane trafficking mediating sorting and transport to lysosomes do not require receptor phosphorylation.     

Adenovirus-mediated p21((WAF1/SDII/CIP1)) gene transfer induces apoptosis of human cervical cancer cell lines

p21((WAF1/SDII/CIP1)) (p21) arrests cell growth by inhibiting cyclin-depend kinases. To explore the potential of using p21 for the gene therapy of cervical cancer, we infected human papillomavirus (HPV)-positive cervical cancer cells (HeLa, SiHa, and Z172) and HPV-negative cervical cancer cells (C33A) with recombinant adenovirus encoding p21 cDNA. The results revealed that effective inhibition of cell growth could be achieved by sense p21 adenovirus but not antisense p21 adenovirus infection and occurred through apoptosis as measured by DNA fragmentation and chromatin condensation. Apoptosis was also observed in xenografts of human cervical cancer cells infected with sense p21 adenovirus, as confirmed by in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). The apoptosis was not prevented by overexpression of the bcl-2 transgene. To sum up, the apoptotic effect suggests that p21 should be a tumoricidal agent instead of a tumoristatic agent in preventing cervical cancers. In addition, our report substantiates the combination of the high efficiency of adenovirus vector-mediated gene delivery and the apoptotic effect of p21.     

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.     

Making sense of lung-cancer gene-expression profiles

A report on the Critical Assessment of Microarray Data Analysis (CAMDA'03) meeting and competition, Durham, USA, 12-14 November 2003.     

Combinatorial synthesis of substituted 3-(2-indolyl)piperidines and 2-phenyl indoles as inhibitors of ZipA-FtsZ interaction

The ZipA-FtsZ protein-protein interaction is a potential target for antibacterial therapy. The design and parallel synthesis of a combinatorial library of small molecules, which target the FtsZ binding area on ZipA are described. Compounds were demonstrated to bind to the FtsZ binding domain of ZipA by HSQC NMR and to inhibit cell division in a cell elongation assay.     

Synthesis and anti-inflammatory evaluation of 4-anilinofuro[2,3-b]quinoline and 4-phenoxyfuro[2,3-b]quinoline derivatives. Part 3

Mast cells, neutrophils and macrophages are important inflammatory cells that have been implicated in the pathogenesis of acute and chronic inflammatory diseases. To explore a novel anti-inflammatory agent, we have synthesized certain 4-anilinofuro[2,3-b]quinoline and 4-phenoxyfuro[2,3-b]quinoline derivatives and evaluated their anti-inflammatory activities by reaction of 3,4-dichlorofuro[2,3-b]quinoline with appropriate Ar-NH(2) or Ar-OH. Compounds 6a and 15 were proved to be more potent than the reference inhibitor, mepacrine for the inhibition of rat peritoneal mast cell degranulation with IC(50) values of 6.5 and 16.4 microM, respectively. Compounds 2b, 6a, 10, and 15 also showed potent inhibitory activity (IC(50)=7.2-29.4 microM) for the secretion of lysosomal enzyme and beta-glucuronidase from neutrophils. These results also indicated that oxime derivatives are more potent than the respective ketone precursors (6a> or =2a; 7a> or =3), and the substituent such as Me at the oxime decreased inhibitory activity (6a> or =6b; 7a> or =7b). Among these derivatives, compound 6a showed the most potent activity with IC(50) values of 6.5-11.6 microM for the inhibition of mast cell degranulation and neutrophil degranulation.     

NOS inhibition accelerates atherogenesis: reversal by exercise

In this study, we assessed the effects of chronic exercise training (12 wk) on atherosclerotic lesion formation in hypercholesterolemic apolipoprotein E-deficient mice (n = 31). At the age of 9 wk, mice were assigned to the following groups: sedentary (Sed; n = 9); exercise (Ex; n = 12); sedentary and oral NG-nitro-L-arginine (L-NNA, Sed-NA; n = 4), or exercise and oral L-NNA (Ex-NA; n = 6). Chronic exercise training was performed on a treadmill for 12 wk (6 times/wk and twice for 1 h/day) at a final speed of 22 m/min, and an 8 degrees grade. L-NNA was discontinued 5 days before final treadmill testing. The farthest distance run to exhaustion was observed in Ex-NA mice (Sed: 306 +/- 32 m; Ex: 640 +/- 87; Sed-NA: 451 +/- 109 m; Ex-NA: 820 +/- 49 m; all P < 0.05). Lesion formation was assessed in the proximal ascending aorta by dissection microscopy after oil red O staining. The aortas of Sed-NA mice manifested a threefold increase in lesion formation compared with the other groups. This L-NNA-induced lesion formation was reduced by chronic exercise training (Sed, 786 +/- 144; Ex, 780 +/- 206; Sed-NA, 2,147 +/- 522; Ex-NA, 851 +/- 253; Sed-NA vs. all other groups: P < 0.001). In conclusion, treatment with oral L-NNA (an nitric oxide synthase antagonist) leads to accelerated atherogenesis in genetically determined hypercholesterolemic mice. This adverse effect can be overcome by chronic exercise training.     

Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity. Different oligomers activate different signal transduction pathways

Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-kappaB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-alpha at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state.     

Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein

The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese encephalitis virus (JEV) envelope protein. The JEV domain III forms a beta-barrel type structure composed of six antiparallel beta-strands resembling the immunoglobulin constant domain. We have also identified epitopes of the JEV domain III to its neutralizing antibody by chemical shift perturbation measurements. Site-directed mutagenesis experiments are performed to confirm the NMR results. Our study provides a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses.