Andrew Keightley

Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway.

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.

Synthesis and optical properties of triphenylene-based dendritic donor perylene diimide acceptor systems

A donor-acceptor charge transfer system based on two discotic mesogens has been synthesized. The donor is either a triphenylene (POG0) or a triphenylene-based conjugated dendron (POG1), while the acceptor is a perylene diimide (PDI) core. The donors are covalently linked to the bay positions of the PDI core through an ether linkage. In chloroform, due to the short donor-acceptor distance and the matching frontier orbital levels, photoinduced charge transfer from either the donor excitation or the acceptor excitation are both thermodynamically and kinetically favored, resulting in efficient quenching of both donor and acceptor fluorescence. In a less polar solvent, hexane, while charge transfer is still the dominant mechanism for decay of the excited electronic state of POG1, photoinduced charge transfer is no longer energetically favorable for POG0 when the acceptor PDI core is excited, making the PDI core of POG0 weakly fluorescent in chloroform but strongly so in hexane. In solid film, POG0 is highly aggregated through both PDI-PDI and triphenylene-triphenylene homotopic stacking. POG1, on the other hand, aggregates through triphenylene dendrons with limited PDI-PDI core stacking, presumably due to the steric hindrance caused by bulky triphenylene moieties which block the access to the PDI core. The efficient photoinduced charge transfer, coupled with the homotopic stacking that forms separated electron-transporting PDI-stacked columns and hole transporting triphenylene-stacked columns, suggests that the reported donor-acceptor systems based on dual-discotic mesogens are potentially new efficient photovoltaic materials.

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

Noncanonical FK506-binding protein BDBT binds DBT to enhance its circadian function and forms foci at night.

The kinase DOUBLETIME is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of DOUBLETIME interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knockdown of CG17282 produced behavioral arrhythmicity and long periods and high levels of hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME. Overexpression of DOUBLETIME in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances DOUBLETIME-dependent PERIOD degradation, indicating that CG17282 stimulates DOUBLETIMEs circadian function. In photoreceptors, CG17282 accumulates rhythmically in PERIOD- and DOUBLETIME-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds DOUBLETIME and tetratricopeptide repeats that may promote assembly of larger protein complexes. We have named CG17282 BRIDE OF DOUBLETIME and established it as a mediator of DOUBLETIMEs effects on PERIOD, most likely in cytosolic foci that regulate PERIOD nuclear accumulation.

The role of casein kinase I in the Drosophila circadian clock.

The circadian clock mechanism in organisms as diverse as cyanobacteria and humans involves both transcriptional and posttranslational regulation of key clock components. One of the roles for the posttranslational regulation is to time the degradation of the targeted clock proteins, so that their oscillation profiles are out of phase with respect to those of the mRNAs from which they are translated. In Drosophila, the circadian transcriptional regulator PERIOD (PER) is targeted for degradation by a kinase (DOUBLETIME or DBT) orthologous to mammalian kinases (CKIɛ and CKIδ) that also target mammalian PER. Since these kinases are not regulated by second messengers, the mechanism (if any) for their regulation is not known. We are investigating the possibility that regulation of DBT is conferred by other proteins that associate with DBT and PER. In this chapter, the methods we are employing to identify and analyze these factors are discussed. These methods include expression of wild type and mutant proteins with the GAL4/UAS binary expression approach, analysis of DBT in Drosophila S2 cells, in vitro kinase assays with DBT isolated from S2 cells, and proteomic analysis of DBT-containing complexes and of DBT phosphorylation with mass spectrometry. The work has led to the discovery of a previously unrecognized circadian rhythm component (Bride of DBT, a noncanonical FK506-binding protein) and the mapping of autophosphorylation sites within the DBT C-terminal domain with potential regulatory roles.

Expression and purification of full length mouse metal response element binding transcription factor-1 using Pichia pastoris

The metal response element binding transcription factor-1 (MTF-1) is an important stress response, heavy metal detoxification, and zinc homeostasis factor in eukaryotic organisms from Drosophila to humans. MTF-1 transcriptional regulation is primarily mediated by elevated levels of labile zinc, which direct MTF-1 to bind the metal response element (MRE). This process involves direct zinc binding to the MTF-1 zinc fingers, and zinc dependent interaction of the MTF-1 acidic region with the p300 coactivator protein. Here, the first recombinant expression system for mutant and wild type (WT) mouse MTF-1 (mMTF-1) suitable for biochemical and biophysical studies in vitro is reported. Using the methyltropic yeast Pichia pastoris, nearly half-milligram recombinant WT and mutant mMTF-1 were produced per liter of P. pastoris cell culture, and purified by a FLAG-tag epitope. Using a first pass ammonium sulfate purification, followed by anti-FLAG affinity resin, mMTF-1 was purified to >95% purity. This recombinant mMTF-1 was then assayed for direct protein-protein interactions with p300 by co-immunoprecipitation. Surface plasmon resonance studies on mMTF-1 provided the first quantitative DNA binding affinity measurements to the MRE promotor element (K(d)=5±3 nM). Both assays demonstrated the functional activity of the recombinant mMTF-1, while elucidating the molecular basis for mMTF-1-p300 functional synergy, and provided new insights into the mMTF-1 domain specific roles in DNA binding. Overall, this production system provides accessibility for the first time to a multitude of in vitro studies using recombinant mutant and WT mMTF-1, which greatly facilitates new approaches to understanding the complex and varied functions of this protein.