Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Classical anticytokinins do not interact with cytokinin receptors but inhibit cyclin-dependent kinases

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.     

A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor

The molecular chaperone HEAT SHOCK PROTEIN90 (HSP90) is essential for the maturation of key regulatory proteins in eukaryotes and for the response to temperature stress. Earlier, we have reported that fungi living in association with plants of the Sonoran desert produce small molecule inhibitors of mammalian HSP90. Here, we address whether elaboration of the HSP90 inhibitor monocillin I (MON) by the rhizosphere fungus Paraphaeosphaeria quadriseptata affects plant HSP90 and plant environmental responsiveness. We demonstrate that MON binds Arabidopsis (Arabidopsis thaliana) HSP90 and can inhibit the function of HSP90 in lysates of wheat (Triticum aestivum) germ. MON treatment of Arabidopsis seedlings induced HSP101 and HSP70, conserved components of the stress response. Application of MON, or growth in the presence of MON, allowed Arabidopsis wild type but not AtHSP101 knockout mutant seedlings to survive otherwise lethal temperature stress. Finally, cocultivation of P. quadriseptata with Arabidopsis enhanced plant heat stress tolerance. These data demonstrate that HSP90-inhibitory compounds produced by fungi can influence plant growth and responses to the environment.     

Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly

The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.     

Functional insights from structural genomics

Structural genomics efforts have produced structural information, either directly or by modeling, for thousands of proteins over the past few years. While many of these proteins have known functions, a large percentage of them have not been characterized at the functional level. The structural information has provided valuable functional insights on some of these proteins, through careful structural analyses, serendipity, and structure-guided functional screening. Some of the success stories based on structures solved at the Northeast Structural Genomics Consortium (NESG) are reported here. These include a novel methyl salicylate esterase with important role in plant innate immunity, a novel RNA methyltransferase (H. influenzae yggJ (HI0303)), a novel spermidine/spermine N-acetyltransferase (B. subtilis PaiA), a novel methyltransferase or AdoMet binding protein (A. fulgidus AF_0241), an ATP:cob(I)alamin adenosyltransferase (B. subtilis YvqK), a novel carboxysome pore (E. coli EutN), a proline racemase homolog with a disrupted active site (B. melitensis BME11586), an FMN-dependent enzyme (S. pneumoniae SP_1951), and a 12-stranded β-barrel with a novel fold (V. parahaemolyticus VPA1032).     

Diatomic ligand discrimination by the heme oxygenases from Neisseria meningitidis and Pseudomonas aeruginosa

Heme oxygenases have an increased binding affinity for O2 relative to CO. Such discrimination is critical to the function of HO enzymes because one of the main products of heme catabolism is CO. Kinetic studies of mammalian and bacterial HO proteins reveal a significant decrease in the dissociation rate of O2 relative to other heme proteins such as myoglobin. Here we report the kinetic rate constants for the binding of O2 and CO by the heme oxygenases from Neisseria meningitidis (nmHO) and Pseudomonas aeruginosa (paHO). A combination of stopped-flow kinetic and laser flash photolysis experiments reveal that nmHO and paHO both maintain a similar degree of ligand discrimination as mammalian HO-1 and the HO from Corynebacterium diphtheriae. However, in addition to the observed decrease in dissociation rate for O2 by both nmHO and paHO, kinetic analyses show an increase in dissociation rate for CO by these two enzymes. The crystal structures of nmHO and paHO both contain significant differences from the mammalian HO-1 and bacterial C. diphtheriae HO structures, which suggests a structural basis for ligand discrimination in nmHO and paHO.     

Refinement of the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers

We present here the parmbsc0 force field, a refinement of the AMBER parm99 force field, where emphasis has been made on the correct representation of the alpha/gamma concerted rotation in nucleic acids (NAs). The modified force field corrects overpopulations of the alpha/gamma = (g+,t) backbone that were seen in long (more than 10 ns) simulations with previous AMBER parameter sets (parm94-99). The force field has been derived by fitting to high-level quantum mechanical data and verified by comparison with very high-level quantum mechanical calculations and by a very extensive comparison between simulations and experimental data. The set of validation simulations includes two of the longest trajectories published to date for the DNA duplex (200 ns each) and the largest variety of NA structures studied to date (15 different NA families and 97 individual structures). The total simulation time used to validate the force field includes near 1 mus of state-of-the-art molecular dynamics simulations in aqueous solution.