Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17?

The yeast serine threonine kinase Atg1 appears to be a key regulator of autophagy and its kinase activity is crucial for autophagy induction. Recent reports have indicated that a mammalian Atg1 homolog, UNC-51-like kinase (ULK) 1, is required for autophagy. We found that ULK1 localizes to the autophagic isolation membrane and its kinase activity is important for autophagy induction. Furthermore, we identified a focal adhesion kinase (FAK) family interacting protein of 200 kD (FIP200) as a ULK-interacting protein. FIP200 also localizes to the isolation membrane together with ULK. Using FIP200-deficient cells, we found that FIP200 is essential for autophagosome formation and the proper function of ULK. Here, we discuss the role of the ULK-FIP200 complex in autophagy and the possibility that FIP200 functions as a mammalian counterpart of Atg17.     

Boron deficiency: How does the defect in cell wall damage the cells?

Boron (B) is an essential micronutrient for vascular plants. Boron plays a structural role in cell walls through binding to pectic polysaccharides. It still remains unclear how B deficiency, and hence probably alterations in cell wall structure, leads to various metabolic disorders and cell death. To understand the process, we analyzed the physiological changes in suspension- cultured tobacco (Nicotiana tabacum) BY-2 cells under B deficiency. The results indicated that the cells deprived of B did not undergo a typical programmed cell death process. Oxidative damage was proven to be the direct and major cause of cell death. We discuss possible mechanisms for the generation and accumulation of reactive oxygen species under B deprivation.Key words: boron deficiency, cell death, cell wall, oxidative damage, pectic polysaccharides, rhamnogalacturonan II, tobaccoBoron (B) deficiency is the most widespread micronutrient deficiency around the world and causes large losses in crop production both quantitatively and qualitatively.¹ Boron deficiency affects vegetative and reproductive growth of plants resulting in inhibition of cell expansion, death of meristem and reduced fertility.²Plants contain B both in a water-soluble and insoluble form. In intact plants, the amount of water-soluble B fluctuates with the quantity of B supplied, while insoluble B does not.³ The appearance of B deficiency symptoms coincides with the decrease of water-insoluble B, from which it is concluded that the insoluble B is the functional form while the soluble B represents the surplus. We found at least 98% of the insoluble B in tobacco cells bound to the cell wall,⁴ and identified their molecular entity as the borate diester with rhamnogalacturonan II (RG-II) regions of pectic polysaccharides.⁵ The diester crosslinks pectic polysaccharides to form a network and thereby contributes to construction of a supramolecular cell wall structure.⁶ Mutant plants with altered RG-II structures are dwarf and sterile, indicating that the B-RG-II complex is essential for normal plant growth and development.⁷ Increasing evidence indicates that B is also essential for animals.⁸ The requirement for B in organisms lacking cell walls implies that B may also have additional roles in plants. To date, however, no molecule other than apiosyl residues in pectic polysaccharides has been demonstrated to form a borate ester which could be stable enough under physiological conditions. Thus it is reasonable to consider that B functions primarily, if not exclusively, as a structural component of the cell wall, and B deficiency symptoms arise from disturbance of the cell wall structure. How, then, does the disturbed cell wall structure lead to the damage and cell death that are observed under B deficiency? To understand the linkage, we have analyzed physiological changes of suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells under B deficiency.When cells at the log phase of growth were transferred to B-free media, cell death was detectable as early as 12 h after the treatment. As cell walls play pivotal roles in plant development and growth, we assumed that the B deprivation, which probably causes aberrant cell wall structure, might induce programmed cell death (PCD) as an active response to eliminate damaged cells. Then we examined if the known biochemical hallmark of PCD could be observed in cells deprived of B (hereafter referred to as -B cells). However, internucleosomal DNA fragmentation, decrease in antioxidant content and antioxidant enzyme expression,⁹ or protection from death by cycloheximide, were not detected in these cells, suggesting that the cell death is necrosis. We found oxidative damage to be the direct and major cause of cell death, because -B cells contained more reactive oxygen species (ROS) than control cells, and because cell death was effectively suppressed by supplementing the media with lipophilic antioxidants. The deprivation treatment did not induce an oxidative burst, as the extracellular H₂O₂ concentration was not significantly different between -B and control cells at all time points examined. Resupply of B immediately suppressed cell death. Collectively, these results suggest that low but persistent ROS production occurred under the -B condition.In the study described above, we demonstrated that B deprivation, and hence probably a defective cell wall structure, leads to oxidative damage. How and why B deprivation induces ROS overproduction remains to be clarified. We hypothesize that ROS are originally produced as a signal for disturbance of the cell wall structure, and build up to a toxic level unless B is resupplied and the cell wall structure is restored. It has been reported that the mechanical strength of the squash root cell wall decreases within minutes after B deprivation.^10,11 The mechanical change could be brought about by insufficient crosslinking of pectic polysaccharides at RG-II regions, as the B-RG-II complex significantly contributes to the wall tensile strength.¹² If the cell wall becomes weaker and less resistant to turgor, then the plasma membrane would stretch. The change may lead to opening of mechanosensitive channels¹³ and generation of signals for the altered cell wall structure. To test this hypothesis, we are now analyzing the immediate and early responses of tobacco BY-2 cells to B deprivation, and preliminary results do indicate the involvement of Ca²⁺ influx in the responses. Identification of the mechanism by which cells sense the external B status will greatly contribute to our understanding of the cell wall-symplast interaction in plants.¹⁴     

Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels

Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, we identified WASP-interacting protein homologue (WIP)-1 in Caenorhabditis elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and wsp-1(RNAi) treatment decreased levels of WIP-1 protein. However, wsp-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in wsp-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for wsp-1. Immunostaining showed that WIP-1 was expressed by migrating hypodermal cells, as was WSP-1. This expression during ventral enclosure was reduced in wip-1(RNAi)-treated embryos and wsp-1(RNAi)-treated embryos. Our results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1.     

Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity

The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.     

Tti1 and Tel2 Are Critical Factors in Mammalian Target of Rapamycin Complex Assembly

Mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family and is a major regulator of translation, cell growth, and autophagy. mTOR exists in two distinct complexes, mTORC1 and mTORC2, that differ in their subunit composition. In this study, we identified KIAA0406 as a novel mTOR-interacting protein. Because it has sequence homology with Schizosaccharomyces pombe Tti1, we named it mammalian Tti1. Tti1 constitutively interacts with mTOR in both mTORC1 and mTORC2. Knockdown of Tti1 suppresses phosphorylation of both mTORC1 substrates (S6K1 and 4E-BP1) and an mTORC2 substrate (Akt) and also induces autophagy. S. pombe Tti1 binds to Tel2, a protein whose mammalian homolog was recently reported to regulate the stability of PIKKs. We confirmed that Tti1 binds to Tel2 also in mammalian cells, and Tti1 interacts with and stabilizes all six members of the PIKK family of proteins (mTOR, ATM, ATR, DNA-PKcs, SMG-1, and TRRAP). Furthermore, using immunoprecipitation and size-exclusion chromatography analyses, we found that knockdown of either Tti1 or Tel2 causes disassembly of mTORC1 and mTORC2. These results indicate that Tti1 and Tel2 are important not only for mTOR stability but also for assembly of the mTOR complexes to maintain their activities.     

Phospholipase C-related but Catalytically Inactive Protein Is Required for Insulin-induced Cell Surface Expression of ��-Aminobutyric Acid Type A Receptors

The γ-aminobutyric acid type A (GABA_A) receptors play a pivotal role in fast synaptic inhibition in the central nervous system. One of the key factors for determining synaptic strength is the number of receptors on the postsynaptic membrane, which is maintained by the balance between cell surface insertion and endocytosis of the receptors. In this study, we investigated whether phospholipase C-related but catalytically inactive protein (PRIP) is involved in insulin-induced GABA_A receptor insertion. Insulin potentiated the GABA-induced Cl⁻ current (I_GABA) by about 30% in wild-type neurons, but not in PRIP1 and PRIP2 double-knock-out (DKO) neurons, suggesting that PRIP is involved in insulin-induced potentiation. The phosphorylation level of the GABA_A receptor β-subunit was increased by about 30% in the wild-type neurons but not in the mutant neurons, which were similar to the changes observed in I_GABA. We also revealed that PRIP recruited active Akt to the GABA_A receptors by forming a ternary complex under insulin stimulation. The disruption of the binding between PRIP and the GABA_A receptor β-subunit by PRIP interference peptide attenuated the insulin potentiation of I_GABA. Taken together, these results suggest that PRIP is involved in insulin-induced GABA_A receptor insertion by recruiting active Akt to the receptor complex.     

Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9

Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state.Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.     

Toxic isolectins from the mushroom Boletus venenatus

Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS–PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galβ1 → 4GlcNAcβ1 → 4Manβ1 → 4GlcNAcβ1 → 4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats.     

Up-regulation of per mRNA expression by parathyroid hormone through a protein kinase A-CREB-dependent mechanism in chondrocytes

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h in both cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5 cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage.     

Electrolyzed Reduced Water Supplemented with Platinum Nanoparticles Suppresses Promotion of Two-stage Cell Transformation

In the two-stage cell transformation theory, cancer cells first receive initiation, which is mainly caused by DNA damage, and then promotion, which enhances transformation. Murine Balb/c 3T3 cells are widely used for transformation experiments because they lose contact inhibition ability when transformed. Electrolyzed reduced water (ERW), which is produced near a cathode during electrolysis of water, is an alkaline drinking water that is beneficial to health. ERW contains a high concentration of dissolved hydrogen and scavenge reactive oxygen species (ROS), along with a small amount of platinum (Pt) nanoparticles (Pt nps) derived from Pt-coated titanium electrodes. Pt nps stably disperse in aqueous solution for a long time, and convert hydrogen molecules to active hydrogen (atomic hydrogen) that can scavenge ROS. Therefore, ERW supplemented with synthesized Pt nps is a model strong reduced water. This is the first report that ERW supplemented with synthesized Pt nps strongly prevents transformation of Balb/c 3T3 cells. ERW was prepared by electrolysis of 0.002 M NaOH solution using a batch-type electrolysis device. Balb/c 3T3 cells were treated with 3-methyl cholanthrene (MCA) as an initiation substance, followed by treatment with phorbol-12-myristate-13-acetate (PMA) as a promotion substance. MCA/PMA-induced formation of a transformation focus was strongly suppressed by ERW supplemented with Pt nps but not by ERW or Pt nps individually. ERW supplemented with Pt nps suppressed transformation at the promoter stage, not at initiation, suggesting that ERW supplemented with Pt nps suppressed the PMA-induced augmentation of intracellular ROS. ERW supplemented with Pt nps is a potential new antioxidant against carcinogenesis.