Cycloheximide inhibits starvation-induced autophagy through mTORC1 activation

Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.     

Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.Both constitutive endocytosis and activated endocytosis are highly regulated events by which cells take up nutrients and internalize receptors for recycling or degradation (47). Endocytosed molecules are delivered to early endosomes, where the components are sorted to the cell surface for recycling back to the plasma membrane, or to late endosomes to be degraded in lysosomes (17). The molecular mechanisms regulating these events are not fully understood.One of the major protein families involved in the trafficking of membrane compartments is sorting nexins (SNXs), which are characterized by the presence of phox homology (PX) domains (8, 65). The PX domain is a protein module which consists of approximately 130 amino acids with three β-strands followed by three α-helices forming a helical subdomain, and the general function of this module is to interact with the head groups of inositol phospholipids through which parental proteins are targeted to specific cellular compartments. Most of the SNXs examined to date specifically recognize phosphatidylinositol 3-phosphate [PtdIns(3)P], which is found predominately in early endosomes (11). The founding member of the SNX family, SNX1, was initially identified as an interaction partner of epidermal growth factor receptor (EGFR), and the expression of SNX1 enhanced lysosomal degradation of EGFR (38); therefore, SNXs are most likely to be involved in the trafficking of many different families of receptors which are recycled to the cell surface or sent to the lysosome for degradation (19). On the other hand, PX domain-containing proteins have also been reported to bind to phosphoinositides other than PtdIns(3)P and to have functions independent of receptor trafficking (54). For example, phospholipase D is a PX domain-containing protein that hydrolyzes phosphatidylcholine to produce a second-messenger molecule, phosphatidic acid. Interestingly, phospholipase D has been recently shown to accelerate EGFR endocytosis by activating dynamin GTPase through its PX domain but independently of lipase activity (39). Cytokine-independent survival kinase (CISK) is a PX domain-containing protein kinase that has also been shown to regulate sorting of a chemokine receptor CXCR4 through AIP4, the CXCR4 ubiquitin ligase (60). RGS-PX1, a GTPase-activating protein for Gαs of heterotrimeric GTP-binding proteins, and KIF16B, a PX domain-containing kinesin superfamily member, have been shown to regulate EGFR trafficking (27, 72) and are now grouped into the SNX family as SNX13 and SNX26, respectively.Another feature of the PX domain is a well-conserved polyproline sequence (PXXP) in the variable loop between α1 and α2 helices, which led to the original identification of the PX domain as a SH3 domain-binding partner (53). The physiological importance of both intermolecular and intramolecular interactions mediated by polyproline sequences has been shown in various molecules, including phospholipase D2 (33) and p47^phox (1). In mammals, there are currently 47 proteins harboring PX domains, and 30 proteins are termed SNXs (59). The functions of these proteins have just begun to be revealed.Actin cytoskeletal dynamics have been implicated not only in cell motility and cytokinesis but also in endocytic processes, although the necessity and role in endocytosis in higher eukaryotic cells remain ambiguous (12, 34, 35, 55). The WASP homology 2 (WH2) domain is known as an actin-binding motif found in regulators of the actin cytoskeleton, including Wiskott-Aldrich syndrome protein (WASP), Scar/WASP-family verprolin-homologous protein (WAVE), verprolin/WASP-interacting protein (WIP), missing in metastasis (MIM), and β-thymosins (52). Some proteins with WH2 domains, such as β-thymosin, prevent actin filament assembly by sequestering actin monomers, while others, such as N-WASP and the Drosophila protein Ciboulot participate in barbed-end actin assembly (52). Recently, the structural basis for these opposite functions of WH2 domains was demonstrated; the interaction of the C-terminal region of β-thymosin/WH2 domain with the pointed end of the actin monomer accounts for the switch in function from inhibition to promotion of actin assembly (26). WH2 domains exist in almost 20 proteins, whose functions remain to be clarified.In the present study, we isolated a new multimodular protein (termed PXK), conserved in multicellular organisms including humans through flies, which possesses a PX domain, a protein kinase-like domain, and a WH2 domain. We show that the PX and WH2 domains function as PtdIns(3)P and actin-binding domains, respectively. PXK expression in COS cells accelerated ligand-induced EGFR endocytosis and degradation that was dependent on a functional PX domain but independent of the WH2 domain. PXK also enhanced ubiquitination of EGFR induced by EGF stimulation in these cells. Based on these results, we propose that PXK is a functional sorting nexin that may play an additional role in cellular function via its interaction with the actin cytoskeleton.     

GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51–DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.     

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.     

Characterization of thermostable native alkaline phosphatase from an aerobic hyperthermophilic archaeon, <Emphasis Type="Italic">Aeropyrum pernix</Emphasis> K1

This paper reports the characterization of an alkaline phosphatase (AP) from an aerobic hyperthermophilic Archaeon Aeropyrum pernix K1. The native AP was purified into homogeneity. The enzyme is predicted as a homodimeric structure with a native molecular mass of about 75 kDa and monomer of about 40 kDa. Apparent optimum pH and temperature were estimated at 10.0 and above 95°C, respectively. Magnesium ion increased both the stability and the activity of the enzyme. A. pernix AP has been demonstrated as a very thermostable AP, retaining about 76% of its activity after being incubated at 90°C for 5.5 h and 67% of its activity after being incubated at 100°C for 2.5 h, respectively, under the presence of Mg(II). Enzyme activity was increased in addition of exogenous Mg(II), Ca(II), Zn(II), and Co(II).     

CD43 collaborates with P-selectin glycoprotein ligand-1 to mediate E-selectin-dependent T cell migration into inflamed skin

Activated T cell migration into nonlymphoid tissues is initiated by the interactions of P- and E-selectin expressed on endothelial cells and their ligands on T cells. P-selectin glycoprotein ligand-1 (PSGL-1) has been the only E-selectin ligand demonstrated to function during the in vivo migration of activated T cells. We show in this study that CD43-deficient Th1 cells, like PSGL-1-deficient cells, exhibited reduced E-selectin-binding activity compared with wild-type cells. Th1 cells with a PSGL-1 and CD43 double deficiency showed even less E-selectin-binding activity. In migration assays in which adoptively transferred cells migrate to inflamed skin P- and E-selectin dependently, CD43 contributed significantly to PSGL-1-independent Th1 cell migration. In addition, in vivo activated T cells from the draining lymph nodes of sensitized mice deficient in PSGL-1 and/or CD43 showed significantly decreased E-selectin-binding activity and migration efficiency, with T cells from double-deficient mice showing the most profound decrease. Collectively, these results demonstrate that the CD43 expressed on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.     

Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 microm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the "final" cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation.