The formin-binding protein 17, FBP17, binds via a TNKS binding motif to tankyrase, a protein involved in telomere maintenance

In acute myelogenous and lymphoid leukemias, rearrangements involving the MLL (mixed lineage leukemia) gene at chromosome 11q23 are frequent. The truncated MLL protein is fused in-frame to a series of partner proteins. We previously identified the formin-binding protein 17 (FBP17) as such an MLL fusion partner. In this study, we explored in vivo physiological interaction partners of FBP17 using a two-hybrid assay and found tankyrase (TNKS), an ADP-ribose polymerase protein involved in telomere maintenance and mitogen-activated protein kinase signaling. We demonstrate that FBP17 binds via a special TNKS-binding motif to tankyrase. The physiological relevance is indicated by co-immunoprecipitation of endogenous proteins in 293T cells.     

Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking

Tubules and vesicles are membrane carriers involved in traffic along the endocytic and secretory routes. The small GTPase Arf6 regulates a recycling branch of short dynamic tubular intermediates used by major histocompatibility class I (MHC-I) molecules to traffic through vesicles between endosomes and the plasma membrane. We observed that Arf6 also affects a second network of very long and stable tubules containing MHC-I, many of which correspond to deep invaginations of the plasma membrane. Treatment with wortmannin, an inhibitor of phosphatidylinositol-3-phosphate kinase, prevents formation of the short dynamic tubules while increasing the number of the long and very stable ones. Expression of NefAAAA, a mutant form of HIV Nef, increases the number of cells containing the stable tubules, and is used here as a tool to facilitate their study. Photoactivation of NefAAAA-PA-GFP demonstrates that this molecule traffics from endosomes to the tubules. Finally, live-cell imaging also shows internalization of MHC-I molecules into these tubules, suggesting that this is an additional route for MHC-I traffic.     

Misfolding of the prion protein at the plasma membrane induces endocytosis, intracellular retention and degradation

Suramin induces misfolding of the cellular prion protein (PrP(C)) and interferes with the propagation of infectious scrapie prions. A mechanistic analysis of this effect revealed that suramin-induced misfolding occurs at the plasma membrane and is dependent on the proximal region of the C-terminal domain (aa 90-158) of PrP(C). The conformational transition induces rapid internalization, mediated by the unstructured N-terminal domain, and subsequent intracellular degradation of PrP(C). As a consequence, PrP Delta N adopts a misfolded conformation at the plasma membrane; however, internalization is significantly delayed. We also found that misfolding and intracellular retention of PrP(C) can be induced by copper and that, moreover, copper interferes with the propagation of the pathogenic prion protein (PrP(Sc)) in scrapie-infected N2a cells. Our study revealed a quality control pathway for aberrant PrP conformers present at the plasma membrane and identified distinct PrP domains involved.     

A novel small molecule,LAS-0811, inhibits alcohol-induced apoptosis in VL-17A cells

One of the pathways by which alcohol induces hepatocyte apoptosis is via oxidative stress. We screened several chemically-synthesized small molecules and found LAS-0811, which inhibits oxidative stress. In this study, we elucidated its role in inhibiting alcohol-induced apoptosis in hepatocyte-like VL-17A cells. VL-17A cells were pre-incubated with LAS-0811, followed by ethanol incubation. Ethanol-induced reactive oxygen species and apoptosis were significantly inhibited in LAS-0811 pre-treated cells. VL-17A cells were transfected with a reporter (ARE/TK-GFP) plasmid containing green fluorescent protein (GFP) as a reporter gene and the anti-oxidant response element as the promoter. LAS-0811 pre-treatment significantly induced the GFP expression compared to the cells treated with ethanol alone. LAS-0811 induced the activation of nrf2 and enhanced the expression and activity of glutathione peroxidase, one of the downstream targets of nrf2. The results indicate that LAS-0811 protects VL-17A cells against ethanol-induced oxidative stress and apoptosis at least in part via nrf2 activation.     

Occurrence of unusual tubular invaginations of the plasma membrane in smooth muscle cells of the lamprey,Lampetra japonica

Numerous tubular structures were observed in the surface region of smooth muscle cells making up the vascular walls in the lamprey, Lampetra japonica; they were designated as surface tubules. The limiting membrane of the surface tubules was connected to the plasma membrane, allowing communication of the lumen of the tubule with the extracellular space. Tannic acid reacted with osmium, serving as an extracellular marker, penetrated into the tubules but not into the intracellular organelles, such as the endoplasmic reticulum and the Golgi complex. The surface tubules were grouped in longitudinal parallel rows, separated from each other by tubule-free areas where dense plaques were present. Each tubule was fairly cylindrical (approximately 60 nm in diameter) and often ramified into two or three branches with a blind end. Occasionally, these tubules were encircled by the sarcoplasmic reticulum which was located immediately beneath the plasma membrane. Similar tubules were also observed in the surface region of vascular endothelial cells and fibroblasts in the adventitial connective tissue. The possibility that the surface tubules in the present observations are analogous to the smooth muscle caveolae or the striated muscle T-tubule is discussed.     

Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex

In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase. Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex. Expression of Hakai in epithelial cells disrupts cell--cell contacts and enhances endocytosis of E-cadherin and cell motility. Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial--mesenchymal transitions in development or metastasis.     

A novel protein, MUDENG, induces cell death in cytotoxic T cells

A screening system comprised of a randomized hybrid-ribozyme library has previously been used to identify pro-death genes in Fas-mediated apoptosis, and short sequence information of candidate genes from this system was previously reported by Kawasaki and Taira [H. Kawasaki, K. Taira, A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries, Nucleic Acids Res. 30 (2002) 3609-3614]. In this study, we have cloned the full-length of the candidate’s open reading frames and found that one of the candidates, referred to as MUDENG (Mu-2 related death-inducing gene), which is composed of 490 amino acids that contain the adaptin domain found in the μ2 subunit of APs related to clathrin-mediated endocytosis, is able to induce cell death by itself. Ectopic expression of MUDENG induced cell death in Jurkat T cells and HeLa cells. In addition, when MUDENG expression was evaluated by immnuohistochemical staining, it was found in most tissues, including the intestine and testis. Furthermore, MUDENG appears to be evolutionary conserved from mammals to amphibians, suggesting that it may have a common role in cell death. Taken together, these results suggest that MUDENG is likely to play an important role in cell death in various tissues.     

A novel protein, Romo1, induces ROS production in the mitochondria

The majority of endogenous reactive oxygen species (ROS) are produced in the mitochondrial respiratory chain. An imbalance in ROS production alters the intracellular redox homeostasis, triggers DNA damage, and contributes to cancer development and progression. This study identified a novel protein, reactive oxygen species modulator 1 (Romo1), which is localized in the mitochondria. Romo1 was found to increase the level of ROS in the cells. Increased Romo1 expression was observed in various cancer cell lines. This suggests that the increased Romo1 expression during cancer progression may cause persistent oxidative stress to tumor cells, which can increase their malignancy.     

Autophagonizer, a novel synthetic small molecule, induces autophagic cell death

Autophagy is an apoptosis-independent mechanism of cell death that protects the cell from environmental imbalances and infection by pathogens. We identified a novel small molecule, 2-(3-Benzyl-4-oxo-3,4,5,6,7,8-hexahydro-benzo[4,5]thieno[2,3-d]pyrimidin-2-ylsulfanylmethyl)-oxazole-4-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (referred as autophagonizer), using high-content cell-based screening and the autophagosome marker EGFP-LC3. Autophagonizer inhibited growth and induced cell death in the human tumor cell lines MCF7, HeLa, HCT116, A549, AGS, and HT1080 via a caspase-independent pathway. Conversion of cytosolic LC3-I to autophagosome-associated LC3-II was greatly enhanced by autophagonizer treatment. Transmission electron microscopy and acridine orange staining revealed increased autophagy in the cytoplasm of autophagonizer-treated cells. In conclusion, autophagonizer is a novel autophagy inducer with unique structure, which induces autophagic cell death in the human tumor cell lines.     

Cross-linking of plasmalemmal cholesterol in lymphocytes induces capping, membrane shedding, and endocytosis through coated pits.

By use of a nicked and biotinylated perfringolysin O (BCtheta), which binds to cholesterol specifically, we studied consequences of cross-linking cholesterol in lymphocytes. When bound with BCtheta and then with labeled avidin or streptavidin, capping occurred in most cells within 30 min at 37 degrees C. It was inhibited by cytochalasin D or NaN3, but not by nocodazole. When BCtheta-cholesterol was capped, Thy-1 and transferrin receptor, a GPI-anchored protein and a transmembrane protein, respectively, remained evenly distributed. By fluorescence and electron microscopy, a cluster of small vesicles bound with BCtheta were observed in the cap. They were then shed in the medium or internalized through coated pits. The result indicates that cross-linking of cholesterol in lymphocytes induces capping, but does not affect distribution of membrane proteins, and that the capped cholesterol molecules are either shed as vesicles or endocytosed.     

A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells

Angiotensin II stimulates cellular hypertrophy in cultured vascular smooth muscle and renal proximal tubular cells. This effect is believed to be one of earliest morphological changes of heart and renal failure. However, the precise molecular mechanism involved in angiotensin II-induced hypertrophy is poorly understood. In the present study we report the isolation of a novel angiotensin II type 1 receptor-associated protein. It encodes a 531-amino acid protein. Its mRNA is detected in all human tissues examined but highly expressed in the human kidney, pancreas, heart, and human embryonic kidney cells as well as rat vascular smooth muscle and renal proximal tubular cells. Protein synthesis and relative cell size analyzed by flow cytometry studies indicate that overexpression of the novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in cultured rat vascular smooth muscle and renal proximal tubular cells. In contrast, the hypertrophic effects was reversed in renal proximal tubular cell lines expressing the novel gene in the antisense orientation and its dominant negative mutant, which lacks the last 101 amino acids in its carboxyl-terminal tail. The hypertrophic effects are at least in part mediated via protein kinase B activation or cyclin-dependent kinase inhibitor, p27(kip1) protein expression level in vascular smooth muscle, and renal proximal tubular cells. Moreover, angiotensin II could not stimulate cellular hypertrophy in renal proximal tubular cells expressing the novel gene in the antisense orientation and its mutant. These findings may provide new molecular mechanisms to understand hypertrophic agents such as angiotensin II-induced cellular hypertrophy.     

Rab5-activating protein 6, a novel endosomal protein with a role in endocytosis

Rab GTPases are regulators of membrane trafficking that cycle between active (GTP-bound) and inactive (GDP-bound) states. In this study, we report the identification of a new human Rab5 guanine nucleotide exchange factor (GEF), which we have named RAP6 (Rab5-activating protein 6). RAP6 contains a Rab5 GEF and a Ras GAP domain. We show that the Vps9 domain is sufficient for the interaction of RAP6 with GDP-bound Rab5 and that RAP6 stimulates Rab5 guanine nucleotide exchange. We also find that the Ras GAP domain of RAP6 shows GAP activity for Ras. Immunofluorescence experiments reveal that RAP6 is associated with plasma membrane and small intracellular vesicles that also contain Rab5. Additionally, the overexpression of RAP6 affects both fluid phase and receptor-mediated endocytosis. This study is the first to show that RAP6 is a novel regulator of endocytosis that exhibits GEF activity specific for Rab5 and GAP activity specific for Ras.     

A novel c-Myc-responsive gene, JPO1, participates in neoplastic transformation

We have identified a novel c-Myc-responsive gene, named JPO1, by representational difference analysis. JPO1 responds to two inducible c-Myc systems and behaves as a direct c-Myc target gene. JPO1 mRNA expression is readily detectable in the thymus, small intestine, and colon, whereas expression is relatively low in spleen, bone marrow, and peripheral leukocytes. We cloned a full-length JPO1 cDNA that encodes a 47-kDa nuclear protein. To determine the role of JPO1 in Myc-mediated cellular phenotypes, stable Rat1a fibroblasts overexpressing JPO1 were tested and compared with transformed Rat1a-Myc cells. Although JPO1 has a diminished transforming activity as compared with c-Myc, JPO1 complements a transformation-defective Myc Box II mutant in the Rat1a transformation assay. This complementation provides evidence for a genetic link between c-Myc and JPO1. Similar to c-Myc, JPO1 overexpression enhances the clonogenicity of CB33 human lymphoblastoid cells in methylcellulose assays. These observations suggest that JPO1 participates in c-Myc-mediated transformation, supporting an emerging concept that c-Myc target genes constitute nodal points in a network of pathways that lead from c-Myc to various Myc-related phenotypes and ultimately to tumorigenesis.     

A water molecule participates in the secondary structure of hyaluronan.

The structure of hyaluronan was investigated in water/dimethyl sulphoxide mixtures by using high-field n.m.r. and space-filling molecular models. The secondary structure previously established in detail in 'dry' dimethyl sulphoxide [Heatley, Scott & Hull (1984) Biochem. J. 220, 197-205] undergoes changes on addition of water, compatible with the incorporation of a water bridge between the uronate carboxylate and acetamido NH groups. Molecular models show that such a configuration is highly probable, and saturation-transfer experiments yield rates of NH proton exchange that support this proposed structure. The existence of two distinct stable configurations for hyaluronan, in water-rich and water-poor conditions respectively, may have biological implications, e.g. during its biosynthesis in cell membranes. There are extensive hydrophobic regions in both forms, which may be important for interactions with e.g., membranes, proteins and itself.     

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex.SummaryIn this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.     

Origin of invaginations and vacuoles in liposomes under the effect of endocytosis inducers including oxidants

The action of various substances on the morphology of multilayer membranes made of lipids of egg yolk was studied. It has been shown that electroneutral and anionic compounds scaresly affected liposomes, whereas substances with pronounced basic properties, i.e. peroxidase, hemoglobin, cytochrome c, and RNAase, as well as lanthanium ions, induced the formation of invaginations, vesicles and aggregation of liposomes. Metals with variable valency: Cu2+, Cu4+, Ru6+, including lipid oxidants Fe3+ and UO+, produced similar morphological changes more intensely and moreover destroyed liposomal membranes. The activator of lipid peroxidation, namely ascorbic acid, intensified while antioxidizers such as alpha-tocopherylacetate and ionol removed the action of Fe3+ on liposomes. A protective effect was displayed by Ca2+ and Mg2+ ions and due to an increase in pH medium. Since many tested substances with the basic properties stimulate endocytosis of cells the processes of lipid peroxidation and electrostatic interactions are supposed to be part of endocytosis mechanism which does not involve the metabolic energy. It is also assumed that endocytosis may arise at the stage of protocells in terms of evolutions.     

Microbial alkaloid staurosporine induces formation of nanometer-wide membrane tubular extensions (cytonemes,membrane tethers) in human neutrophils

In the present work, we demonstrate that microbial alkaloid staurosporine (STS) and Ro 31-8220, structurally related to STS protein kinase C inhibitor, caused development of membrane tubular extensions in human neutrophils upon adhesion to fibronectin-coated substrata. STS-induced tubular extensions interconnected neutrophils in a network and bound serum-opsonized bacteria Salmonella enterica serovar Typhimurium. The diameter of STS-induced extensions varied in the range 160-200 nm. The extensions were filled with cytoplasm and covered with membrane, as they included fluorescent cytoplasmic and lipid dyes. Neither protein kinase C inhibitors H-7 and bisindolylmaleimide VII, nor tyrosine protein kinase inhibitors tyrphostin AG 82 and genistein caused such extensions formation. Supposedly, STS induces membrane tubular extension formation promoting actin cytoskeleton depolymerization or affecting NO synthesis.     

Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes

Gap junctions are composed of a family of structural proteins called connexins, which oligomerize into intercellular channels and function to exchange low molecular weight metabolites and ions between adjacent cells. We have cloned a new member of the connexin family from lens cDNA, with a predicted molecular mass of 46 kD, called rat connexin46 (Cx46). Since a full-length cDNA corresponding to the 2.8-kb mRNA was not obtained, the stop codon and surrounding sequences were confirmed from rat genomic DNA. The RNA coding for this protein is abundant in lens fibers and detectable in both myocardium and kidney. Western analysis of both rat and bovine lens membrane proteins, using the anti-MP70 monoclonal antibody 6-4-B2-C6 and three anti-peptide antibodies against Cx46 demonstrates that Cx46 and MP70 are different proteins. Immunocytochemistry demonstrates that both proteins are localized in the same lens fiber junctional maculae. Synthesis of Cx46 in either reticulocyte lysate or Xenopus oocytes yields a 46-kD polypeptide; all anti-Cx46 antisera recognize a protein in rat lens membranes 5-10 kD larger, suggesting substantive lenticular posttranslational processing of the native translation product. Oocytes that have synthesized Cx46 depolarize and lyse within 24 h, a phenomenon never observed after expression of rat connexins 32 or 43 (Cx32 and Cx43). Lysis is prevented by osmotically buffering the oocytes with 5% Ficoll. Ficoll-buffered oocytes expressing Cx46 are permeable to Lucifer Yellow but not FITC-labeled BSA, indicating the presence of selective membrane permeabilities. Cx43-expressing oocytes are impermeable to Lucifer Yellow. Voltage-gated whole cell currents are measured in oocytes injected with dilute concentrations of Cx46 but not Cx43 mRNA. These currents are activated at potentials positive to -10 mV. Unlike other connexins expressed in Xenopus oocytes, these results suggest that unprocessed Cx46 induces nonselective channels in the oolemma that are voltage dependent and opened by large depolarizations.     

A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation–reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c . 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c . 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.