RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway.

Removal of damaged mitochondria is orchestrated by a pathway involving the PINK1 kinase and the PARKIN ubiquitin ligase. Ubiquitin chains assembled by PARKIN on the mitochondrial outer membrane recruit autophagy cargo receptors in complexes with TBK1 protein kinase. While TBK1 is known to phosphorylate cargo receptors to promote ubiquitin binding, it is unknown whether TBK1 phosphorylates other proteins to promote mitophagy. Using global quantitative proteomics, we identified S72 in RAB7A, a RAB previously linked with mitophagy, as a dynamic target of TBK1 upon mitochondrial depolarization. TBK1 directly phosphorylates RAB7AS72, but not several other RABs known to be phosphorylated on the homologous residue by LRRK2, in vitro, and this modification requires PARKIN activity in vivo. Interaction proteomics using nonphosphorylatable and phosphomimetic RAB7A mutants revealed loss of association of RAB7AS72E with RAB GDP dissociation inhibitor and increased association with the DENN domain-containing heterodimer FLCN-FNIP1. FLCN-FNIP1 is recruited to damaged mitochondria, and this process is inhibited in cells expressing RAB7AS72A. Moreover, nonphosphorylatable RAB7A failed to support efficient mitophagy, as well as recruitment of ATG9A-positive vesicles to damaged mitochondria. These data reveal a novel function for TBK1 in mitophagy, which parallels that of LRRK2-mediated phosphorylation of the homologous site in distinct RABs to control membrane trafficking.

Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening.

Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based short hairpin RNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.

Partition behavior of surfactants, butanol, and salt during application of density-modified displacement of dense non-aqueous phase liquids.

Density-modified displacement (DMD) is a recent approach for removal of trapped dense NAPL (DNAPL). In this study, butanol and surfactant are contacted with the DNAPL to both reduce the density as well as release the trapped DNAPL (perchloroethylene: PCE). The objective of the study was to determine the distribution of each component (e.g., butanol, surfactant, water, PCE) between the original aqueous and PCE phases during the application of DMD. The results indicated that the presence of the surfactant increased the amount of n-butanol required to make the NAPL phase reach its desired density. In addition, water and anionic surfactant were found to partition along with the BuOH into the PCE phase. The water also found partitioned to reverse micelles in the modified phase. Addition of salt was seen to increase partitioning of surfactant to BuOH containing PCE phase. Subsequently, a large amount of water was solubilized into reverse micelles which lead to significantly increase in volume of the PCE phase. This work thus demonstrates the role of each component and the implications for the operation design of an aquifer treatment using the DMD technique.

Solubilization of dibutyltin dichloride with surfactant solutions in single and mixed oil systems.

The harmful effects of organometallic compounds and their metabolites on the environment and human health require the development of more effective remediation methods. Surfactant enhanced remediation has been considered as a potential method for the removal of organometallic compounds; however, additional understanding is needed about the solubilization processes of these compounds. The surfactant enhanced solubilization of dibutyltin dichloride (DBT), an organometallic compound, was the focus of this research. In addition, the synergistic effects of DBT solubilization in perchloroethylene (PCE) and decane mixtures were evaluated. The results indicate that PCE and decane were solubilized into the core of these surfactant micelles in both single and mixed oil systems. DBT solubilization was limited when DBT alone was present (single oil system), and the nature of the solubilization isotherm suggests that DBT solubilization tended to occur near the micelle surface in a single oil system. DBT solubilization was found to increase when present in the PCE and decane oil mixture. PCE and decane may have facilitated the solubilization of DBT because they were solubilized in the micelle core. From this study, it may be concluded that the DBT behaves like polar oil such as dodecanol, having properties of a polar organic compound.

Hydrophilic-lipophilic deviation (HLD) method for characterizing conventional and extended surfactants.

An accurate determination of the hydrophilic-lipophilic nature of surfactants plays an essential role in guiding the formulation of microemulsion with the goal of achieving low interfacial tension (IFT) and high solubilization. While several empirical models have been proposed as simple tools for predicting surfactant characteristics and microemulsion conditions, only a few of these models are fundamentally based yet convenient to use. In this work, the hydrophilic-lipophilic deviation (HLD) approach was used with mixed surfactant systems to determine the surfactant characteristic (sigma) and the sigmaK parameter of conventional and extended surfactants. To our knowledge, this is the first time that the HLD index has been used to represent the hydrophilic-lipophilic behavior of extended surfactants. It was observed that inserting PO and/or EO groups in extended surfactants play a key role in altering sigma values and sigmaK values. Finally, the sigma parameters found in this work were combined with the HLD equation and used to demonstrate its practical utility for guiding the optimum formulation (in this case, optimum salinity) for a microemulsion system.

Effect of admicellar properties on adsolubilization: column studies and solute transport.

Mixtures of anionic and cationic surfactants exhibit synergistic behavior as evidenced by low critical micelle concentrations (CMC) of the mixed system, increased surface activity, and improved detergency performance. The adsorption of a single-head anionic surfactant, sodium dodecyl sulfate (SDS), in mixture with a twin-head cationic surfactant, pentamethyl-octadecyl-1,3-propane diammonium dichloride (PODD), showed synergism of adsorption onto silica when present at a mixing ratio of 1:3 (cationic-rich), and also demonstrated lower surfactant desorption with water flushing of columns packed with the surfactant-modified media. In addition, the proportion of the mixed surfactants in the admicelles moved from the initial ratio of 1:3 towards equimolar after rinsing the surfactant-modified silica absorbent. The retardation of organic solutes passing through columns packed with modified-silica adsorbent increased nominally three fold for silica modified with mixed surfactants versus single surfactants (retardation factors increase from 4.0 to 12.8 for styrene and from 32.1 to 90.2 for ethylcyclohexane for single and mixed surfactants, respectively). Thus, this study demonstrates that mixed surfactant systems more effectively modified the silica surface than single surfactant systems both in terms of enhanced retardation of organic solutes and in terms of reduced surfactant desorption.

Stress and mTORture signaling.

The TOR (target of rapamycin) pathway is an evolutionarily conserved signaling module regulating cell growth (accumulation of mass) in response to a variety of environmental cues such as nutrient availability, hypoxia, DNA damage and osmotic stress. Its pivotal role in cellular and organismal homeostasis is reflected in the fact that unrestrained signaling activity in mammals is associated with the occurrence of disease states including inflammation, cancer and diabetes. The existence of TOR homologs in unicellular organisms whose growth is affected by environmental factors, such as temperature, nutrients and osmolarity, suggests an ancient role for the TOR signaling network in the surveillance of stress conditions. Here, we will summarize recent advances in the TOR signaling field with special emphasis on how stress conditions impinge on insulin/insulin-like growth factor signaling/TOR signaling.

Adsorption of the quinolone antibiotic nalidixic acid onto anion-exchange and neutral polymers.

Pharmaceutical products are being found in surface and ground waters around the world. While the consequences to human health are unknown, it is suspected that these contaminants adversely alter aquatic ecosystems. This study presents adsorption results for one pharmaceutical product, nalidixic acid (NA), on neutral and anion-exchange polymers at three pH values. The adsorption of NA below and above its pKa of approximately 6 on polymers with different matrices, forms, and degrees of polarity were evaluated. By comparing the Freundlich constants KF and n, the results show that the neutral form of NA adsorbs to a greater extent on neutral polymers, and that the anionic form of NA adsorbs more to anion-exchange polymers. Also, results of the adsorption experiments suggest that aromatic ring interactions between NA and the surface of both neutral and anion-exchange polymers are important in the adsorption process. These results have important implications for the treatment of pharmaceutical-contaminated water, as many pharmaceutical contaminants are ionizable and have aromatic rings in their structure. This study illustrates the importance of pH and sorbate and sorbent structure in considering polymer sorption for treatment of pharmaceutical-contaminated aqueous systems.

Raptor and mTOR: subunits of a nutrient-sensitive complex.

mTOR/RAFT1/FRAP is the target of the FKBP12-rapamycin complex as well as a central component of a nutrient- and hormone-sensitive pathway that controls cellular growth. Recent work reveals that mTOR interacts with a novel evolutionarily conserved protein that we named raptor, for "regulatory associated protein of mTOR." Raptor has several roles in the mTOR pathway. It is necessary for nutrient-mediated activation of the downstream effector S6K1 and increases in cell size. In addition, under conditions that repress the mTOR pathway, the association of raptor with mTOR is strengthened, leading to a decrease in mTOR kinase activity. Raptor is a critical component of the mTOR pathway that regulates cell growth in response to nutrient levels by associating with mTOR.

Simultaneous uptake of anionic surfactants and micellar-solubilized contaminants using anion-exchange resins.

This research studied simultaneous uptake of anionic surfactants and micellar-solubilized organic contaminants by anion-exchange resins. Anionic surfactant molecules adsorbed onto the positively charged resin mainly through electrostatic attraction, while the micellar-solubilized contaminants were excluded from aqueous solutions once the remaining micelles could no longer solubilize them. Data suggest that the excess contaminants adsorbed onto the resin skeleton and admicelle layer formed on the resin surface through hydrophobic interactions and eventually partitioned into the resin gel phase matrix. In batch adsorption, the contaminant solubilization capacity did not decrease linearly with respect to surfactant concentration decrease due to the increased solution counterion activity during anion exchange, and caused "delayed" contaminant uptake relative to that of the surfactant. No such effect occurred in continuous column adsorption, where the surfactant and contaminant breakthrough occurred simultaneously. Surfactant head and tail group properties, along with resin structure and particle size significantly affected surfactant and contaminant uptake rates. Relative to recovering the surfactant, the high exchange potential of the anionic surfactant prevented effective surfactant desorption, even at high electrolyte concentration and in the presence of a cosolvent. The resin matrix also had high affinity for the partitioned contaminant, and the contaminant elution from the resin seemed to be controlled by equilibrium partitioning.

FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse.

The FKBP-12-rapamycin associated protein (FRAP, also known as mTOR and RAFT-1) is a member of the phosphoinositide kinase related kinase family. FRAP has serine/threonine kinase activity and mediates the cellular response to mitogens through signaling to p70s6 kinase (p70(s6k)) and 4E-BP1, resulting in an increase in translation of subsets of cellular mRNAs. Translational up-regulation is blocked by inactivation of FRAP signaling by rapamycin, resulting in G(1) cell cycle arrest. Rapamycin is used as an immunosuppressant for kidney transplants and is currently under investigation as an antiproliferative agent in tumors because of its ability to block FRAP activity. Although the role of FRAP has been extensively studied in vitro, characterization of mammalian FRAP function in vivo has been limited to the immune system and tumor models. Here we report the identification of a loss-of-function mutation in the mouse FRAP gene, which illustrates a requirement for FRAP activity in embryonic development. Our studies also determined that rapamycin treatment of the early embryo results in a phenotype indistinguishable from the FRAP mutant, demonstrating that rapamycin has teratogenic activity.

Solvent extraction for separating micellar-solubilized contaminants and anionic surfactants.

Decontamination of contaminant-laden surfactant solutions is critical to successful implementation of surfactant-enhanced aquifer remediation (SEAR). Solvent extraction was studied for removing micellar-solubilized contaminants having low equivalent alkyl carbon numbers (EACNs) from surfactant solutions. Factors influencing the solvent extraction of micellar-solubilized contaminant were studied, including surfactant concentration, solution salinity, solvent solubilization, and solvent/solution volumetric ratio. A model was developed to quantify the impacts of these factors on contaminant removal. The good agreement between experimental results and model predictions corroborates assumptions made in the model development. From these results, it is concluded that extracting solvents must have much higher EACNs than that of the contaminant to reduce the micellar solubilization of the solvents, which can significantly reduce contaminant removal efficiency. However, the highest EACN solvent is not necessarily the best one for contaminant removal due to other constraints (e.g., molecular weight and viscosity). Increasing the total surfactant concentration or salinity of an anionic surfactant solution increases its contaminant solubilization capacity but reduces the contaminant removal efficiency by solvent extraction. Continuous column extraction operated at a low column surface loading rate allowed contaminant partitioning to approach equilibrium conditions, and multistage column extraction was able to improve the contaminant removal efficiency while minimizing solvent requirement.

Diabetes mellitus and exocrine pancreatic dysfunction in perk-/- mice reveals a role for translational control in secretory cell survival.

The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. PERK is highly expressed in mouse pancreas, an organ active in protein secretion. Under physiological conditions, PERK was partially activated, accounting for much of the phosphorylated eIF2alpha in the pancreas. The exocrine and endocrine pancreas developed normally in Perk-/- mice. Postnatally, ER distention and activation of the ER stress transducer IRE1alpha accompanied increased cell death and led to progressive diabetes mellitus and exocrine pancreatic insufficiency. These findings suggest a special role for translational control in protecting secretory cells from ER stress.

Microarrays of cells expressing defined cDNAs.

Genome and expressed sequence tag projects are rapidly cataloguing and cloning the genes of higher organisms, including humans. An emerging challenge is to rapidly uncover the functions of genes and to identify gene products with desired properties. We have developed a microarray-driven gene expression system for the functional analysis of many gene products in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of non-transfected cells. By printing sets of complementary DNAs cloned in expression vectors, we make microarrays whose features are clusters of live cells that express a defined cDNA at each location. Here we demonstrate two uses for our approach: as an alternative to protein microarrays for the identification of drug targets, and as an expression cloning system for the discovery of gene products that alter cellular physiology. By screening transfected cell microarrays expressing 192 different cDNAs, we identified proteins involved in tyrosine kinase signalling, apoptosis and cell adhesion, and with distinct subcellular distributions.

Model coupling intraparticle diffusion/sorption, nonlinear sorption, and biodegradation processes.

Diffusion, sorption and biodegradation are key processes impacting the efficiency of natural attenuation. While each process has been studied individually, limited information exists on the kinetic coupling of these processes. In this paper, a model is presented that couples nonlinear and nonequilibrium sorption (intraparticle diffusion) with biodegradation kinetics. Initially, these processes are studied independently (i.e., intraparticle diffusion, nonlinear sorption and biodegradation), with appropriate parameters determined from these independent studies. Then, the coupled processes are studied, with an initial data set used to determine biodegradation constants that were subsequently used to successfully predict the behavior of a second data set. The validated model is then used to conduct a sensitivity analysis, which reveals conditions where biodegradation becomes desorption rate-limited. If the chemical is not pre-equilibrated with the soil prior to the onset of biodegradation, then fast sorption will reduce aqueous concentrations and thus biodegradation rates. Another sensitivity analysis demonstrates the importance of including nonlinear sorption in a coupled diffusion/sorption and biodegradation model. While predictions based on linear sorption isotherms agree well with solution concentrations, for the conditions evaluated this approach overestimates the percentage of contaminant biodegraded by as much as 50%. This research demonstrates that nonlinear sorption should be coupled with diffusion/sorption and biodegradation models in order to accurately predict bioremediation and natural attenuation processes. To our knowledge this study is unique in studying nonlinear sorption coupled with intraparticle diffusion and biodegradation kinetics with natural media.

Impacts of heterogeneous organic matter on phenanthrene sorption: different soil and sediment samples.

Organic petrography has been proposed as a tool for characterizing the heterogeneous organic matter present in soil and sediment samples. A new simplified method is proposed as a quantitative means of interpreting observed sorption behavior for phenanthrene and different soils and sediments based on their organic petrographical characterization. This method is tested under singe solute conditions and at phenanthrene concentration of 1 microg/L. Since the opaque organic matter fraction dominates the sorption process, we propose that by quantifying this fraction one can interpret organic content normalized sorption distribution coefficient (Koc) values for a sample. While this method was developed and tested for various samples within the same aquifer, in the current study the method is validated for soil and sediment samples from different sites that cover a wide range of organic matter origin, age, and organic content. All 10 soil and sediment samples studied had log Koc values for the opaque particles between 5.6 and 6.8. This range of Koc values illustrates the heterogeneity of opaque particles between sites and geological formations and thus the need to characterize the opaque fraction of materials on a site-by-site basis.

Localization of ribophorin II to the endoplasmic reticulum involves both its transmembrane and cytoplasmic domains.

Proteins that are concentrated in specific compartments of the endomembrane system in order to exert their organelle-specific function must possess specific localization signals that prevent their transport to distal regions of the exocytic pathway. Some resident proteins of the endoplasmic reticulum (ER) that are known to escape with low efficiency from this organelle to a post ER compartment are recognized by a recycling receptor and brought back to their site of residence. Other ER proteins, however, appear to be retained in the ER by mechanisms that operate in the organelle itself. The mammalian oligosaccharyltransferase (OST) is a protein complex that effects the cotranslational N-glycosylation of newly synthesized polypeptides, and is composed of at least four rough ER-specific membrane proteins: ribophorins I and II (RI and RII), OST48, and Dadl. The mechanism(s) by which the subunits of this complex are retained in the ER are not well understood. In an effort to identify the domains within RII responsible for its ER localization we have studied the fate of chimeric proteins in which one or more RII domains were replaced by the corresponding ones of the Tac antigen, the latter being a well characterized plasma membrane protein that lacks intrinsic ER retention signals and serves to provide a neutral framework for the identification of retention signals in other proteins. We found that the luminal domain of RII by itself does not contain retention information, while the cytoplasmic and transmembrane domains contain independent ER localization signals. We also show that the retention function of the transmembrane domain is strengthened by the presence of a flanking luminal region consisting of 15 amino acids.

Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation.

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Here we demonstrate that protein kinase Cdelta (PKCdelta) associates with RAFT1 and that PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation.

In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein.

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.