Interaction of ferredoxin:NADP+ oxidoreductase with phycobilisomes and phycobilisome substructures of the cyanobacterium Synechococcus sp. strain PCC 7002

The enzyme ferredoxin-NADP(+) oxidoreductase (FNR) from Synechococcus sp. PCC 7002 has an extended structure comprising three domains (FNR-3D) (Schluchter, W. M., and Bryant, D. A. (1992) Biochemistry 31, 3092-3102). Phycobilisome (PBS) preparations from wild-type cells contained from 1.0 to 1.6 molecules of FNR-3D per PBS, with an average value of 1.3 FNR per PBS. A maximum of two FNR-3D molecules could be specifically bound to wild-type PBS via the N-terminal, CpcD-like domain of the enzyme when exogenous recombinant FNR-3D (rFNR-3D) was added. To localize the enzyme within the PBS, the interaction of PBS and their substructures with rFNR-3D was further investigated. The binding affinity of rFNR-3D for phycocyanin (PC) hexamers, which contained a 22-kDa proteolytic fragment derived from CpcG, the L(RC)(27) linker polypeptide, was higher than its affinity for PC hexamers containing no linker protein. PBS from a cpcD3 mutant, which lacks the 9-kDa, PC-associated rod linker, incorporated up to six rFNR-3D molecules per PBS. PBS of a cpcC mutant, which has peripheral rods that contain single PC hexamers, also incorporated up to six rFNR-3D molecules per PBS. Direct competition binding experiments showed that PBS from the cpcD3 mutant bound more enzyme than PBS from the cpcC mutant. These observations support the hypothesis that the enzyme binds preferentially to the distal ends of the peripheral rods of the PBS. These data also show that the relative affinity order of the PC complexes for FNR-3D is as follows: (alpha(PC)beta(PC))(6)-L(R)(33) > (alpha(PC)beta(PC))(6)-L(RC)(27) > (alpha(PC)beta(PC))(6). The data suggest that, during the assembly of the PBS, FNR-3D could be displaced to the periphery according to its relative binding affinity for different PC subcomplexes. Thus, FNR-3D would not interfere with the light absorption and energy transfer properties of PC in the peripheral rods of the PBS. The implications of this localization of FNR within the PBS with respect to its function in cyanobacteria are discussed.     

Biophysical characterization of the interactions of HTI-286 with tubulin heterodimer and microtubules

HTI-286 is a synthetic analogue of the natural product hemiasterlin and is a potent antimitotic agent. HTI-286 inhibits the proliferation of tumor cells during mitosis. The observed antimitotic activity is due to the binding of HTI-286 to tubulin. This report details the effects of HTI-286 on soluble tubulin and preassembled microtubules. HTI-286 binds tubulin monomer and oligomerizes it to an 18.5 S species corresponding to a discrete ring structure consisting of about 13 tubulin units as determined by sedimentation equilibrium analyses. The rate of formation of the oligomers is dependent on the concentration of HTI-286 and the time of incubation. Tubulin oligomers, specifically the 18.5 S species, form slowly. The interactions of HTI-286 with tubulin were studied by isothermal titration calorimetry. HTI-286 binds tubulin rapidly, and the initial association of HTI-286 with tubulin is enthalpically driven with a DeltaH value of -14 kcal/mol at 25 degrees C and a dissociation constant of ca. 100 nM. However, the accompanying tubulin oligomerization event does not produce measurable heats at 25 degrees C. The dissociation constant estimated from the changes in the intrinsic fluorescence of tubulin was found to be consistent with the calorimetric results. Both HTI-286 and hemiasterlin bind tubulin with nearly equal potency. However, the stability of the tubulin oligomers is not identical under size-exclusion column chromatographic conditions. The tubulin oligomers formed in the presence of HTI-286 dissociate on the column, while the corresponding oligomers formed in the presence of hemiasterlin are stable. Tubulin undergoes a change in the secondary structure in the presence of HTI-286, which is evidenced by changes in the circular dichroic absorption spectrum of tubulin. In contrast to the microtubule-stabilizing effects of paclitaxel, both HTI-286 and hemiasterlin depolymerize preassembled microtubules at micromolar concentrations.     

Bidirectional electron transfer in photosystem I: electron transfer on the PsaA side is not essential for phototrophic growth in Chlamydomonas

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.     

Role of the extracellular and cytoplasmic domains of CD44 in the rolling interaction of lymphoid cells with hyaluronan under physiologic flow

CD44 can function as an adhesion receptor that mediates leukocyte rolling on hyaluronan (HA). To study the contributions of different domains of the standard isoform of CD44 to cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected with wild type (WT) or mutated cDNA constructs. A parallel flow chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that rolled and the rolling velocity was inversely proportional to the amount of cell surface CD44. When the cytoplasmic domain distal to Gly(305) or sequences that serve as binding sites for cytoskeletal linker proteins, were deleted or replaced with foreign sequences, no significant changes in the rolling behavior of mutant cells, compared with the transfectant expressing CD44WT, were observed. Transfectants lacking 64 amino acids of the cytoplasmic tail distal to Cys(295) adhered to HA but showed enhanced rolling at low shear forces. When 83 amino acids from the "non-conserved" membrane-proximal region of the CD44 extracellular domain were deleted, cells adhered firmly to the HA substrate and did not roll at any fluid shear force tested. Unlike wild type cells that exhibited a nearly homogeneous distribution of CD44 on a smooth cell surface, cells expressing the non-conserved region deletion mutant accumulated CD44 in membrane protrusions. Disruption of the actin cytoskeleton with cytochalasin B precluded the formation of membrane protrusions, however, treated cells still adhered firmly to HA and did not roll. We conclude that interaction between the cytoplasmic domain of CD44 and the cytoskeleton is not required for cell rolling on immobilized ligand. The strong effect of deletion of the non-conserved region of the extracellular domain argues for a critical role of this region in CD44-dependent rolling and adhesion interactions with HA under flow.     

Differential role of actin,clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.     

Mutant analyses define multiple roles for phytochrome C in Arabidopsis photomorphogenesis

The analysis of Arabidopsis mutants deficient in the A, B, D, and E phytochromes has revealed that each of these phytochrome isoforms has both distinct and overlapping roles throughout plant photomorphogenesis. Although overexpression studies of phytochrome C (phyC) have suggested photomorphogenic roles for this receptor, conclusive evidence of function has been lacking as a result of the absence of mutants in the PHYC locus. Here, we describe the isolation of a T-DNA insertion mutant of phyC (phyC-1), the subsequent creation of mutant lines deficient in multiple phytochrome combinations, and the physiological characterization of these lines. In addition to operating as a weak red light sensor, phyC may perform a significant role in the modulation of other photoreceptors. phyA and phyC appear to act redundantly to modulate the phyB-mediated inhibition of hypocotyl elongation in red light and to function together to regulate rosette leaf morphology. In addition, phyC performs a significant role in the modulation of blue light sensing. Several of these phenotypes are supported by the parallel analysis of a quadruple mutant deficient in phytochromes A, B, D, and E, which thus contains only active phyC. Together, these data suggest that phyC has multiple functions throughout plant development that may include working as a coactivator with other phytochromes and the cryptochrome blue light receptors.     

Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.     

Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells

Survivin, a member of inhibitor of apoptosis family of proteins, plays important roles in both cell proliferation and cell death. We previously observed that Survivin is overexpressed in leukemic cell lines and blasts from patients with acute myelogenous leukemia (AML). To understand the roles of Survivin in AML and search for new approaches to the treatment of AML, we inhibited Survivin expression in HL-60 cells with a Survivin anti-sense oligonucleotide (sur-AS-ODN) (ISIS 23722). This blocked significant numbers of HL-60 cells in G2/M phase, and halted cell proliferation at 24 hrs and progressing over time. There was only a slight increase in the number of apoptotic cells at 24 hrs compared with cells treated with nonsense oligonucleotide (NS-ODN). At 48 hrs, however, there were significant increases in sub-G1 phase and annexin V+ cells, suggesting that cell division defects caused cell death. This was supported by the finding that a reduction in the Survivin protein by sur-AS-ODN in cells under serum-free medium did not induce G2/M block and cell death compared to cells treated with NS-ODN. The formation of polyploid cells was observed 48 hrs after sur-AS-ODN treatment, as was the activation of caspase 3, which suggested that apoptotic cell death had occurred. The mitochondrial release of cytochrome C and Smac and the nuclear translocation of the apoptosis-inducing factor were also detected. Our results suggest that Survivin is essential for cell cycle progression in leukemic cells. Reduced Survivin expression causes a cell-cycle defect that leads to cell death through a mitochondrial pathway. This finding has potential utility for therapy of patients with AML.