Regulating cellular trace metal economy in algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blaby-Haas, Crysten E.; Merchant, Sabeeha S.

As indispensable protein cofactors, Fe, Mn, Cu and Zn are at the center of multifaceted acclimation mechanisms that have evolved to ensure extracellular supply meets intracellular demand. In starting with selective transport at the plasma membrane and ending in protein metalation, metal homeostasis in algae involves regulated trafficking of metal ions across membranes, intracellular compartmentalization by proteins and organelles, and metal-sparing/recycling mechanisms to optimize metal-use efficiency. Overlaid on these processes are additional circuits that respond to the metabolic state as well as to the prior metal status of the cell. Here, we focus on recent progress made toward understanding themore » pathways by which the single-celled, green alga Chlamydomonas reinhardtii controls its cellular trace metal economy. We also compare these mechanisms to characterized and putative processes in other algal lineages. Photosynthetic microbes continue to provide insight into cellular regulation and handling of Cu, Fe, Zn and Mn as a function of the nutritional supply and cellular demand for metal cofactors. We found that new experimental tools such as RNA-Seq and subcellular metal imaging are bringing us closer to a molecular understanding of acclimation to supply dynamics in algae and beyond.« less

Regulating cellular trace metal economy in algae

DOE PAGES

Blaby-Haas, Crysten E.; Merchant, Sabeeha S.

2017-06-30

As indispensable protein cofactors, Fe, Mn, Cu and Zn are at the center of multifaceted acclimation mechanisms that have evolved to ensure extracellular supply meets intracellular demand. In starting with selective transport at the plasma membrane and ending in protein metalation, metal homeostasis in algae involves regulated trafficking of metal ions across membranes, intracellular compartmentalization by proteins and organelles, and metal-sparing/recycling mechanisms to optimize metal-use efficiency. Overlaid on these processes are additional circuits that respond to the metabolic state as well as to the prior metal status of the cell. Here, we focus on recent progress made toward understanding themore » pathways by which the single-celled, green alga Chlamydomonas reinhardtii controls its cellular trace metal economy. We also compare these mechanisms to characterized and putative processes in other algal lineages. Photosynthetic microbes continue to provide insight into cellular regulation and handling of Cu, Fe, Zn and Mn as a function of the nutritional supply and cellular demand for metal cofactors. We found that new experimental tools such as RNA-Seq and subcellular metal imaging are bringing us closer to a molecular understanding of acclimation to supply dynamics in algae and beyond.« less

Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

PubMed Central

Hougland, James L.; Gangopadhyay, Soumyashree A.; Fierke, Carol A.

2012-01-01

Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein. PMID:22992747

A Chemical Biology Approach to Interrogate Quorum Sensing Regulated Behaviors at the Molecular and Cellular Level

PubMed Central

Lowery, Colin A.; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan A.; Mee, Jenny M.; Cravatt, Benjamin F.; Miller, Samuel I.; Kaufmann, Gunnar F.; Janda, Kim D.

2013-01-01

SUMMARY Small molecule probes have been employed extensively to explore biological systems and elucidate cellular signaling pathways. In this study, we utilize an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering new processes regulated by AI-2-based quorum sensing (QS), a mechanism of bacterial intracellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intracellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. PMID:23890008

A chemical biology approach to interrogate quorum-sensing regulated behaviors at the molecular and cellular level.

PubMed

Lowery, Colin A; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan; Lively, Jenny M; Cravatt, Benjamin F; Miller, Samuel I; Kaufmann, Gunnar F; Janda, Kim D

2013-07-25

Small molecule probes have been used extensively to explore biologic systems and elucidate cellular signaling pathways. In this study, we use an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering unrecognized processes regulated by AI-2-based quorum-sensing (QS), a mechanism of bacterial intercellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intercellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of uridine 5'-eicosylphosphate as a stimulant of cyclic AMP-dependent cellular function.

PubMed

Yutani, Masahiro; Ogita, Akira; Fujita, Ken-Ichi; Usuki, Yoshinosuke; Tanaka, Toshio

2011-03-01

Sporulation of the yeast Saccharomyces cerevisiae is negatively regulated by cyclic AMP (cAMP). This microbial cell differentiation process was applied for the screening of a substance that can elevate the intracellular cAMP level. Among nucleoside 5'-alkylphosphates, uridine 5'-eicosylphosphate (UMPC20) selectively and predominantly inhibited ascospore formation of the yeast cells. We suppose the inhibitory effect of UMPC20 could indeed reflect the elevation of the cellular cAMP level.

Special Issue on Optochemical and Optogenetic Control of Cellular Processes.

PubMed

Deiters, Alexander

2018-06-06

Diverse optochemical and optobiological approaches are being developed and applied to the light-regulation of cellular processes with exquisite spatial and temporal resolution in cells and multicellular model organisms. In this special issue, experts report some of the latest progress in the expanding field of the optical control of biological systems and present an overview of the state of the art of select approaches. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oxidative stress and protein aggregation during biological aging.

PubMed

Squier, T C

2001-09-01

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation. Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.

Size-Dependent Regulation of Intracellular Trafficking of Polystyrene Nanoparticle-Based Drug-Delivery Systems.

PubMed

Wang, Ting; Wang, Lu; Li, Xiaoming; Hu, Xingjie; Han, Yuping; Luo, Yao; Wang, Zejun; Li, Qian; Aldalbahi, Ali; Wang, Lihua; Song, Shiping; Fan, Chunhai; Zhao, Yun; Wang, Maolin; Chen, Nan

2017-06-07

Nanoparticles (NPs) have shown great promise as intracellular imaging probes or nanocarriers and are increasingly being used in biomedical applications. A detailed understanding of how NPs get "in and out" of cells is important for developing new nanomaterials with improved selectivity and less cytotoxicity. Both physical and chemical characteristics have been proven to regulate the cellular uptake of NPs. However, the exocytosis process and its regulation are less explored. Herein, we investigated the size-regulated endocytosis and exocytosis of carboxylated polystyrene (PS) NPs. PS NPs with a smaller size were endocytosed mainly through the clathrin-dependent pathway, whereas PS NPs with a larger size preferred caveolae-mediated endocytosis. Furthermore, our results revealed exocytosis of larger PS NPs and tracked the dynamic process at the single-particle level. These results indicate that particle size is a key factor for the regulation of intracellular trafficking of NPs and provide new insight into the development of more effective cellular nanocarriers.

Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials

PubMed Central

Autefage, Hélène; Littmann, Elena; Hedegaard, Martin A. B.; Von Erlach, Thomas; O’Donnell, Matthew; Burden, Frank R.; Winkler, David A.; Stevens, Molly M.

2015-01-01

Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells’ global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell–material interactions and suggest alternative research routes for evaluating biomaterials to improve their design. PMID:25831522

Move or Die: the Fate of the Tax Oncoprotein of HTLV-1

PubMed Central

Lodewick, Julie; Lamsoul, Isabelle; Bex, Françoise

2011-01-01

The HTLV-1 Tax protein both activates viral replication and is involved in HTLV-1-mediated transformation of T lymphocytes. The transforming properties of Tax include altering the expression of select cellular genes via activation of cellular pathways and perturbation of both cell cycle control mechanisms and apoptotic signals. The recent discovery that Tax undergoes a hierarchical sequence of posttranslational modifications that control its intracellular localization provides provocative insights into the mechanisms regulating Tax transcriptional and transforming activities. PMID:21994756

Characterization of the Potent, Selective Nrf2 Activator, 3-(Pyridin-3-Ylsulfonyl)-5-(Trifluoromethyl)-2H-Chromen-2-One, in Cellular and In Vivo Models of Pulmonary Oxidative Stress.

PubMed

Yonchuk, John G; Foley, Joseph P; Bolognese, Brian J; Logan, Gregory; Wixted, William E; Kou, Jen-Pyng; Chalupowicz, Diana G; Feldser, Heidi G; Sanchez, Yolanda; Nie, Hong; Callahan, James F; Kerns, Jeffrey K; Podolin, Patricia L

2017-10-01

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2 H -chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant ( tert -butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1 β -induced nuclear factor- κ B translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Involvement of autophagy in T cell biology.

PubMed

Oral, Ozlem; Yedier, Ozlem; Kilic, Seval; Gozuacik, Devrim

2017-01-01

Autophagy is an essential cellular pathway that sequesters various cytoplasmic components, including accumulated proteins, damaged organelles or invading microorganisms and delivers them to lysosomes for degradation. The function of autophagy has been reported in various tissues and systems, including its role in the regulation of cellular immunity. Autophagy plays a fundamental role at various stages of T cell maturation. It regulates the thymocyte selection and the generation of T cell repertoire by presenting intracellular antigens to MHC class molecules. Autophagy is crucial for metabolic regulation of T cells, and therefore supports cell survival and homeostasis, particularly in activated mature T cells. Furthermore, deletion of specific autophagy-related genes induces several immunological alterations including differentiation of activated T cells into regulatory, memory or natural killer T cells. In this review, we emphasize the impact of autophagy on T cell development, activation and differentiation, which is pivotal for the adaptive immune system.

Mitochondrial reactive oxygen species regulate the strength of inhibitory GABA-mediated synaptic transmission

NASA Astrophysics Data System (ADS)

Accardi, Michael V.; Daniels, Bryan A.; Brown, Patricia M. G. E.; Fritschy, Jean-Marc; Tyagarajan, Shiva K.; Bowie, Derek

2014-01-01

Neuronal communication imposes a heavy metabolic burden in maintaining ionic gradients essential for action potential firing and synaptic signalling. Although cellular metabolism is known to regulate excitatory neurotransmission, it is still unclear whether the brain’s energy supply affects inhibitory signalling. Here we show that mitochondrial-derived reactive oxygen species (mROS) regulate the strength of postsynaptic GABAA receptors at inhibitory synapses of cerebellar stellate cells. Inhibition is strengthened through a mechanism that selectively recruits α3-containing GABAA receptors into synapses with no discernible effect on resident α1-containing receptors. Since mROS promotes the emergence of postsynaptic events with unique kinetic properties, we conclude that newly recruited α3-containing GABAA receptors are activated by neurotransmitter released onto discrete postsynaptic sites. Although traditionally associated with oxidative stress in neurodegenerative disease, our data identify mROS as a putative homeostatic signalling molecule coupling cellular metabolism to the strength of inhibitory transmission.

Arctigenin preferentially induces tumor cell death under glucose deprivation by inhibiting cellular energy metabolism.

PubMed

Gu, Yuan; Qi, Chunting; Sun, Xiaoxiao; Ma, Xiuquan; Zhang, Haohao; Hu, Lihong; Yuan, Junying; Yu, Qiang

2012-08-15

Selectively eradicating cancer cells with minimum adverse effects on normal cells is a major challenge in the development of anticancer therapy. We hypothesize that nutrient-limiting conditions frequently encountered by cancer cells in poorly vascularized solid tumors might provide an opportunity for developing selective therapy. In this study, we investigated the function and molecular mechanisms of a natural compound, arctigenin, in regulating tumor cell growth. We demonstrated that arctigenin selectively promoted glucose-starved A549 tumor cells to undergo necrosis by inhibiting mitochondrial respiration. In doing so, arctigenin elevated cellular level of reactive oxygen species (ROS) and blocked cellular energy metabolism in the glucose-starved tumor cells. We also demonstrated that cellular ROS generation was caused by intracellular ATP depletion and played an essential role in the arctigenin-induced tumor cell death under the glucose-limiting condition. Furthermore, we combined arctigenin with the glucose analogue 2-deoxyglucose (2DG) and examined their effects on tumor cell growth. Interestingly, this combination displayed preferential cell-death inducing activity against tumor cells compared to normal cells. Hence, we propose that the combination of arctigenin and 2DG may represent a promising new cancer therapy with minimal normal tissue toxicity. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Rapid aquaporin translocation regulates cellular water flow: mechanism of hypotonicity-induced subcellular localization of aquaporin 1 water channel.

PubMed

Conner, Matthew T; Conner, Alex C; Bland, Charlotte E; Taylor, Luke H J; Brown, James E P; Parri, H Rheinallt; Bill, Roslyn M

2012-03-30

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

Features of cues and processes during chloroplast-mediated retrograde signaling in the alga Chlamydomonas

USDA-ARS?s Scientific Manuscript database

Retrograde signalling is a selective process defined by cues generated in chloroplast/mitochondria which traverse membranes and end up regulating nuclear gene expression and protein synthesis. The coding and encoding of organellar message(s) that alter nuclear gene expression and/or cellular metabo...

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis.

PubMed

Trosko, James E

2016-06-15

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules ("quorum sensing"), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or "connexin" genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision-making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global "metabolic disease" crisis.

Regulation of cellular marker modulated upon irradiation of low power laser light in burn injured mice

NASA Astrophysics Data System (ADS)

Rathnakar, Bharath; Prabhu, Vijendra; Rao, Bola Sadashiva Satish; Chandra, Subhash; Rai, Sharada; Mahato, Krishna Kishore

2016-12-01

The present study intends to understand the importance of cellular marker in tissue regeneration regulated upon irradiation of low power laser light in burn inflicted mice. Under anesthetic conditions, the thermal injury was induced on Swiss albino mice of either sex. Following injury, the animals were randomly divided into three groups; i. e., un-illuminated control, the group treated with 5% Povidone iodine (reference standard) and single exposure of 3 J/cm2 (830 nm). Burn tissue samples from each group were excised at day 6 post burn injury upon euthanization and used for histological and immunohistochemical analysis. Haematoxylin and Eosin (H and E) staining was performed on the selected sections to asses proliferation and angiogenesis at day 6 post-injury. For immunohistochemical analysis, tissue sections from all the three treatment groups on day 6 were stained using specific antibody against Proliferating cell nuclear antigen (PCNA). The results of the histological and immunohistochemical analysis showed improved tissue restoration in animals treated with optimal laser influence as compared to un-illuminated controls. The findings of present study clearly demonstrated the beneficial effects of 830 nm laser in burn wound healing and its influence in regulating the cellular marker.

Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome.

PubMed

Yang, Yi; Qin, Meiling; Bao, Puhua; Xu, Wangchao; Xu, Jin

2017-01-01

Autophagy-lysosomal pathway is a cellular protective system to remove aggregated proteins and damaged organelles. Meanwhile, exosome secretion has emerged as a mode to selectively clear the neurotoxic proteins, such as α-synuclein. Mounting evidence suggests that these two cellular processes are coordinated to facilitate the clearance of toxic cellular waste; however the regulators for the transition between these two processes are unclear. Here we show that SCAMP5, a secretory carrier membrane protein significantly induced in the brains of Huntington's disease patients, is quickly and transiently induced by protein stress and autophagic stimulation, and is regulated by the master autophagy transcriptional regulator TFEB. Ironically, SCAMP5 inhibits autophagy flux by blocking the fusion of autophagosomes and lysosomes. Although autophagy is blocked, SCAMP5 does not cause significant protein aggregation in cells. Instead, it promotes the Golgi fragmentation and stimulates the unconventional secretion of the co-localizing α-synuclein via exosome as an exosome component. Therefore, we have identified SCAMP5 as a novel coordinator of autophagy and exosome secretion, which is induced upon protein stress to channel the efficient clearance of toxic proteins via the exosomes rather than autophagy-lysosomal pathway.

Discovery of novel high potent and cellular active ADC type PTP1B inhibitors with selectivity over TC-PTP via modification interacting with C site.

PubMed

Du, Yongli; Zhang, Yanhui; Ling, Hao; Li, Qunyi; Shen, Jingkang

2018-01-20

PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide template to interact with residues Arg47 and Lys41 in the C site of PTP1B by molecular docking aided design resulted in the discovery of a series of novel high potent and selective inhibitors of PTP1B. The structure activity relationship interacting with the C site of PTP1B was well illustrated. Compounds 8 and 18 were shown to be the high potent and most promising PTP1B inhibitors with cellular activity and great selectivity over the highly homologous TCPTP and other PTPs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Discovery of novel, high potent, ABC type PTP1B inhibitors with TCPTP selectivity and cellular activity.

PubMed

Liu, Peihong; Du, Yongli; Song, Lianhua; Shen, Jingkang; Li, Qunyi

2016-08-08

Protein tyrosine phosphatase 1B (PTP1B) as a key negative regulator of both insulin and leptin receptor pathways has been an attractive therapeutic target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. With the goal of enhancing potency and selectivity of the PTP1B inhibitors, a series of methyl salicylate derivatives as ABC type PTP1B inhibitors (P1-P7) were discovered. More importantly, compound P6 exhibited high potent inhibitory activity (IC50 = 50 nM) for PTP1B with 15-fold selectivity over T-cell PTPase (TCPTP). Further studies on cellular activities revealed that compound P6 could enhance insulin-mediated insulin receptor β (IRβ) phosphorylation and insulin-stimulated glucose uptake. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Quantitative Proteomic Analysis of Duck Ovarian Follicles Infected with Duck Tembusu Virus by Label-Free LC-MS

PubMed Central

Han, Kaikai; Zhao, Dongmin; Liu, Yuzhuo; Liu, Qingtao; Huang, Xinmei; Yang, Jing; An, Fengjiao; Li, Yin

2016-01-01

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. DTMUV infection mainly results in significant decreases in egg production in egg-laying ducks within 1–2 weeks post infection. However, information on the comparative protein expression of host tissues in response to DTMUV infection is limited. In the present study, the cellular protein response to DTMUV infection in duck ovarian follicles was analyzed using nano-flow high-performance liquid chromatography-electrospray tandem mass spectrometry. Quantitative proteomic analysis revealed 131 differentially expressed proteins, among which 53 were up regulated and 78 were down regulated. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and mitochondrial pathway. Some selected proteins that were found to be regulated in DTMUV-infected tissues were screened by quantitative real-time PCR to examine their regulation at the transcriptional level, western blot analysis was used to validate the changes of some selected proteins on translational level. To our knowledge, this study is the first to analyze the proteomic changes in duck ovarian follicles following DTMUV infection. The protein-related information obtained in this study may be useful to understand the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity. PMID:27066001

A Proteomic Study of Brassinosteroid Response in Arabidopsis

PubMed Central

Deng, Zhiping; Zhang, Xin; Tang, Wenqiang; Oses-Prieto, Juan A; Suzuki, Nagi; Gendron, Joshua M; Chen, Huanjing; Guan, Shenheng; Chalkley, Robert J.; Peterman, T. Kaye; Burlingame, Alma L.; Wang, Zhi-Yong

2010-01-01

Summary The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we have performed a proteomic study of BR-regulated proteins in Arabidopsis using two-dimensional difference gel electrophoresis (2-D DIGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR insensitive mutant bri1-116 and BR hypersensitive mutant bzr1-1D identified 5 proteins (PATL1, PATL2, THI1, AtMDAR3 and NADP-ME2) affected by both BR-treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knockout mutants or immunoblotting. Interestingly, about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein- and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore, BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in 2-D DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses. PMID:17848588

Non-AUG translation: a new start for protein synthesis in eukaryotes

PubMed Central

Kearse, Michael G.; Wilusz, Jeremy E.

2017-01-01

Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans-acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states. PMID:28982758

The Regulation of a Post-Translational Peptide Acetyltransferase: Strategies for Selectively Modifying the Biological Activity of Neural and Endocrine Peptides

DTIC Science & Technology

1988-02-01

quantitatively miror pathway. Only two of the enzymes which process 8-endorphin have been firmly identified, peptide acetyltransferase and... quantitatively minor. This implied that perhaps peptide acetyltransferase is not a critical determinant of the bioactivity of B-endorphin in brain. If so...provided us with a more difinitive understanding of the role of processing enzyme regulation in the overall biochemical and cellular response of the

Quantitative Differences in a Single Maternal Factor Determine Survival Probabilities among Drosophila Germ Cells.

PubMed

Slaidina, Maija; Lehmann, Ruth

2017-01-23

Germ cell death occurs in many species [1-3] and has been proposed as a mechanism by which the fittest, strongest, or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. Here, we focus on the mechanisms that regulate germ cell survival during embryonic development in Drosophila. We find that the decision to die is a germ cell-intrinsic process linked to quantitative differences in germ plasm inheritance, such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. We demonstrate that the maternal factor lipid phosphate phosphatase Wunen-2 (Wun2) regulates PGC survival in a dose-dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single-cell level, we propose that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, we demonstrate that Wun2 and p53, another regulator of PGC survival, have opposite yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, we hypothesize that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease.

PubMed

Stürner, Elisabeth; Behl, Christian

2017-01-01

In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 ( BCL-2-associated athanogene 3 ). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer's disease (tau-protein), Huntington's disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

Engineering microbial phenotypes through rewiring of genetic networks

PubMed Central

Rodrigues, Rui T.L.; Lee, Sangjin; Haines, Matthew

2017-01-01

Abstract The ability to program cellular behaviour is a major goal of synthetic biology, with applications in health, agriculture and chemicals production. Despite efforts to build ‘orthogonal’ systems, interactions between engineered genetic circuits and the endogenous regulatory network of a host cell can have a significant impact on desired functionality. We have developed a strategy to rewire the endogenous cellular regulatory network of yeast to enhance compatibility with synthetic protein and metabolite production. We found that introducing novel connections in the cellular regulatory network enabled us to increase the production of heterologous proteins and metabolites. This strategy is demonstrated in yeast strains that show significantly enhanced heterologous protein expression and higher titers of terpenoid production. Specifically, we found that the addition of transcriptional regulation between free radical induced signalling and nitrogen regulation provided robust improvement of protein production. Assessment of rewired networks revealed the importance of key topological features such as high betweenness centrality. The generation of rewired transcriptional networks, selection for specific phenotypes, and analysis of resulting library members is a powerful tool for engineering cellular behavior and may enable improved integration of heterologous protein and metabolite pathways. PMID:28369627

Cyclic Nucleotide Phosphodiesterases: important signaling modulators and therapeutic targets

PubMed Central

Ahmad, Faiyaz; Murata, Taku; Simizu, Kasumi; Degerman, Eva; Maurice, Donald; Manganiello, Vincent

2014-01-01

By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases are critical regulators of their intracellular concentrations and their biological effects. Since these intracellular second messengers control many cellular homeostatic processes, dysregulation of their signals and signaling pathways initiate or modulate pathophysiological pathways related to various disease states, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication, chronic obstructive pulmonary disease, and psoriasis. Alterations in expression of PDEs and PDE-gene mutations (especially mutations in PDE6, PDE8B, PDE11A and PDE4) have been implicated in various diseases and cancer pathologies. PDEs also play important role in formation and function of multi-molecular signaling/regulatory complexes called signalosomes. At specific intracellular locations, individual PDEs, together with pathway-specific signaling molecules, regulators, and effectors, are incorporated into specific signalosomes, where they facilitate and regulate compartmentalization of cyclic nucleotide signaling pathways and specific cellular functions. Currently, only a limited number of PDE inhibitors (PDE3, PDE4, PDE5 inhibitors) are used in clinical practice. Future paths to novel drug discovery include the crystal structure-based design approach, which has resulted in generation of more effective family-selective inhibitors, as well as burgeoning development of strategies to alter compartmentalized cyclic nucleotide signaling pathways by selectively targeting individual PDEs and their signalosome partners. PMID:25056711

A proposed role for selective autophagy in regulating auxin-dependent lateral root development under phosphate starvation in Arabidopsis.

PubMed

Sankaranarayanan, Subramanian; Samuel, Marcus A

2015-01-01

Plants respond to limited soil nutrient availability by inducing more lateral roots (LR) to increase the root surface area. At the cellular level, nutrient starvation triggers the process of autophagy through which bulk degradation of cellular materials is achieved to facilitate nutrient mobilization. Whether there is any link between the cellular autophagy and induction of LR had remained unknown. We recently showed that the S-Domain receptor Kinase (ARK2) and U Box/Armadillo Repeat-Containing E3 ligase (PUB9) module is required for lateral root formation under phosphate starvation in Arabidopsis thaliana.(1) We also showed that PUB9 localized to autophagic bodies following either activation by ARK2 or under phosphate starvation and ark2-1/pub9-1 plants displayed lateral root defects with inability to accumulate auxin in the root tips under phosphate starvation.(1) Supplementing exogenous auxin was sufficient to rescue the LR defects in ark2-1/pub9-1 mutant. Blocking of autophagic responses in wild-type Arabidopsis also resulted in inhibition of both lateral roots and auxin accumulation in the root tips indicating the importance of autophagy in mediating auxin accumulation under phosphate starved conditions.(1) Here, we propose a model for ARK2/AtPUB9 module in regulation of lateral root development via selective autophagy.

Signal transduction networks in rheumatoid arthritis

PubMed Central

Hammaker, D; Sweeney, S; Firestein, G

2003-01-01

Signal transduction pathways regulate cellular responses to stress and play a critical role in inflammation. The complexity and specificity of signalling mechanisms represent major hurdles for developing effective, safe therapeutic interventions that target specific molecules. One approach is to dissect the pathways methodically to determine their hierarchy in various cell types and diseases. This approach contributed to the identification and prioritisation of specific kinases that regulate NF-κB and the mitogen activated protein (MAP) kinase cascade as especially attractive targets. Although significant issues remain with regard to the discovery of truly selective kinase inhibitors, the risks that accompany inhibition of fundamental signal transduction mechanisms can potentially be decreased by careful dissection of the pathways and rational target selection. PMID:14532158

The Deep Thioredoxome in Chlamydomonas reinhardtii: New Insights into Redox Regulation.

PubMed

Pérez-Pérez, María Esther; Mauriès, Adeline; Maes, Alexandre; Tourasse, Nicolas J; Hamon, Marion; Lemaire, Stéphane D; Marchand, Christophe H

2017-08-07

Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular processes. This study broadens not only the redox regulation to new enzymes involved in well-known thioredoxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce (aromatic amino acid biosynthesis, nuclear transport, etc). Moreover, we characterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Translational Regulation in Nutrigenomics12

PubMed Central

Liu, Botao; Qian, Shu-Bing

2011-01-01

The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health. PMID:22332093

Preparing the “Soil”: The Premetastatic Niche

PubMed Central

Kaplan, Rosandra N.; Rafii, Shahin; Lyden, David

2010-01-01

Current focus on cancer metastasis has centered on the intrinsic factors regulating the cell autonomous homing of the tumor cells to the metastatic site. Specific up-regulation of fibronectin and clustering of bone marrow–derived cellular infiltrates coexpressing matrix metalloproteinases in distant tissue sites before tumor cell arrival are proving to be indispensable for the initial stages of metastasis. These bone marrow–derived hematopoietic progenitors that express vascular endothelial growth factor receptor 1 mobilize in response to the unique array of growth factors produced by the primary tumor. Their arrival in distant sites represents early changes in the local microenvironment, termed the “premetastatic niche,” which dictate the pattern of metastatic spread. Focus on the early cellular and molecular events in cancer dissemination and selectivity will likely lead to new approaches to detect and prevent metastasis at its earliest inception. PMID:17145848

microRNA therapies in cancer.

PubMed

Rothschild, Sacha I

2014-01-01

MicroRNAs (miRNAs or miRs) are a family of small non-coding RNA species that have been implicated in the control of many fundamental cellular and physiological processes such as cellular differentiation, proliferation, apoptosis and stem cell maintenance. miRNAs regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation. Some microRNAs have been categorized as "oncomiRs" as opposed to "tumor suppressor miRs" Modulating the miRNA activities may provide exciting opportunities for cancer therapy. This review highlights the latest discovery of miRNAs involved in carcinogenesis as well as the potential applications of miRNA regulations in cancer treatment. Several studies have demonstrated the feasibility of restoring tumor suppressive miRNAs and targeting oncogenic miRNAs for cancer therapy using in vivo model systems.

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis

PubMed Central

Trosko, James E.

2016-01-01

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules (“quorum sensing”), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or “connexin” genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision–making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global “metabolic disease” crisis. PMID:27314399

Expression and activity of the 5'-AMP-activated protein kinase pathway in selected tissues during chicken embryonic development.

USDA-ARS?s Scientific Manuscript database

The 5’-AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase and a key part of a kinase signaling cascade that senses cellular energy status (AMP/ATP ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating m...

The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease

PubMed Central

Stürner, Elisabeth; Behl, Christian

2017-01-01

In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer’s disease (tau-protein), Huntington’s disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate. PMID:28680391

Positive and Negative Regulatory Mechanisms for Fine-Tuning Cellularity and Functions of Medullary Thymic Epithelial Cells.

PubMed

Akiyama, Taishin; Tateishi, Ryosuke; Akiyama, Nobuko; Yoshinaga, Riko; Kobayashi, Tetsuya J

2015-01-01

Self-tolerant T cells and regulatory T cells develop in the thymus. A wide variety of cell-cell interactions in the thymus is required for the differentiation, proliferation, and repertoire selection of T cells. Various secreted and cell surface molecules expressed in thymic epithelial cells (TECs) mediate these processes. Moreover, cytokines expressed by cells of hematopoietic origin regulate the cellularity of TECs. Tumor necrosis factor (TNF) family RANK ligand, lymphotoxin, and CD40 ligand, expressed in T cells and innate lymphoid cells (ILCs), promote the differentiation and proliferation of medullary TECs (mTECs) that play critical roles in the induction of immune tolerance. A recent study suggests that interleukin-22 (IL-22) produced by ILCs promotes regeneration of TECs after irradiation. Intriguingly, tumor growth factor-β and osteoprotegerin limit cellularity of mTECs, thereby attenuating regulatory T cell generation. We will review recent insights into the molecular basis for cell-cell interactions regulating differentiation and proliferation of mTECs and also discuss about a perspective on use of mathematical models for understanding this complicated system.

Genetic variation in resistance of the preimplantation bovine embryo to heat shock.

PubMed

Hansen, Peter J

2014-12-01

Reproduction is among the physiological functions in mammals most susceptible to disruption by hyperthermia. Many of the effects of heat stress on function of the oocyte and embryo involve direct effects of elevated temperature (i.e. heat shock) on cellular function. Mammals limit the effects of heat shock by tightly regulating body temperature. This ability is genetically controlled: lines of domestic animals have been developed with superior ability to regulate body temperature during heat stress. Through experimentation in cattle, it is also evident that there is genetic variation in the resistance of cells to the deleterious effects of elevated temperature. Several breeds that were developed in hot climates, including Bos indicus (Brahman, Gir, Nelore and Sahiwal) and Bos taurus (Romosinuano and Senepol) are more resistant to the effects of elevated temperature on cellular function than breeds that evolved in cooler climates (Angus, Holstein and Jersey). Genetic differences are expressed in the preimplantation embryo by Day 4-5 of development (after embryonic genome activation). It is not clear whether genetic differences are expressed in cells in which transcription is repressed (oocytes >100 µm in diameter or embryos at stages before embryonic genome activation). The molecular basis for cellular thermotolerance has also not been established, although there is some suggestion for involvement of heat shock protein 90 and the insulin-like growth factor 1 system. Given the availability of genomic tools for genetic selection, identification of genes controlling cellular resistance to elevated temperature could be followed by progress in selection for those genes within the populations in which they exist. It could also be possible to introduce genes from thermotolerant breeds into thermally sensitive breeds. The ability to edit the genome makes it possible to design new genes that confer protection of cells from stresses like heat shock.

Comparative MicroRNA Expression Patterns in Fibroblasts after Low and High Doses of Low-LET Radiation Exposure

NASA Technical Reports Server (NTRS)

Maes, Olivier C.; Xu, Suying; Hada, Megumi; Wu, Honglu; Wang, Eugenia

2007-01-01

Exposure to ionizing radiation causes DNA damage to cells, and provokes a plethora of cellular responses controlled by unique gene-directed signaling pathways. MicroRNAs (miRNAs) are small (22-nucleotide), non-coding RNAs which functionally silence gene expression by either degrading the messages or inhibiting translation. Here we investigate radiation-dependent changes in these negative regulators by comparing the expression patterns of all 462 known human miRNAs in fibroblasts, after exposure to low (0.1 Gy) or high (2 Gy) doses of X-rays at 30 min, 2, 6 and 24 hrs post-treatment. The expression patterns of microRNAs after low and high doses of radiation show a similar qualitative down-regulation trend at early (0.5 hr) and late (24 hr) time points, with a quantitatively steeper slope following the 2 Gy exposures. Interestingly, an interruption of this downward trend is observed after the 2 Gy exposure, i.e. a significant up-regulation of microRNAs at 2 hrs, then reverting to the downward trend by 6 hrs; this interruption at the intermediate time point was not observed with the 0.1 Gy exposure. At the early time point (0.5 hr), candidate gene targets of selected down-regulated microRNAs, common to both 0.1 and 2 Gy exposures, were those functioning in chromatin remodeling. Candidate target genes of unique up-regulated microRNAs seen at a 2 hr intermediate time point, after the 2 Gy exposure only, are those involved in cell death signaling. Finally, putative target genes of down-regulated microRNAs seen at the late (24 hr) time point after either doses of radiation are those involved in the up-regulation of DNA repair, cell signaling and homeostasis. Thus we hypothesize that after radiation exposure, microRNAs acting as hub negative regulators for unique signaling pathways needed to be down-regulated so as to de-repress their target genes for the proper cellular responses, including DNA repair and cell maintenance. The unique microRNAs up-regulated at 2 hr after 2 Gy suggest the cellular response to functionally suppress the apoptotic death signaling reflex after exposure to high dose radiation. Further analyses with transcriptome and global proteomic profiling will validate the reciprocal expression of signature microRNAs selected in our radiation-exposed cells, and their candidate target gene families, and test our hypothesis that unique radiation-specific microRNAs are keys in governing signaling responses for damage control of this environmental hazard.

Light-dependent governance of cell shape dimensions in cyanobacteria.

PubMed

Montgomery, Beronda L

2015-01-01

The regulation of cellular dimension is important for the function and survival of cells. Cellular dimensions, such as size and shape, are regulated throughout the life cycle of bacteria and can be adapted in response to environmental changes to fine-tune cellular fitness. Cell size and shape are generally coordinated with cell growth and division. Cytoskeletal regulation of cell shape and cell wall biosynthesis and/or deposition occurs in a range of organisms. Photosynthetic organisms, such as cyanobacteria, particularly exhibit light-dependent regulation of morphogenes and generation of reactive oxygen species and other signals that can impact cellular dimensions. Environmental signals initiate adjustments of cellular dimensions, which may be vitally important for optimizing resource acquisition and utilization or for coupling the cellular dimensions with the regulation of subcellular organization to maintain optimal metabolism. Although the involvement of cytoskeletal components in the regulation of cell shape is widely accepted, the signaling factors that regulate cytoskeletal and other distinct components involved in cell shape control, particularly in response to changes in external light cues, remain to be fully elucidated. In this review, factors impacting the inter-coordination of growth and division, the relationship between the regulation of cellular dimensions and central carbon metabolism, and consideration of the effects of specific environment signals, primarily light, on cell dimensions in cyanobacteria will be discussed. Current knowledge about the molecular bases of the light-dependent regulation of cellular dimensions and cell shape in cyanobacteria will be highlighted.

Deployment of the human immunodeficiency virus type 1 protein arsenal: combating the host to enhance viral transcription and providing targets for therapeutic development

PubMed Central

Dahiya, Satinder; Nonnemacher, Michael R.

2012-01-01

Despite the success of highly active antiretroviral therapy in combating human immunodeficiency virus type 1 (HIV-1) infection, the virus still persists in viral reservoirs, often in a state of transcriptional silence. This review focuses on the HIV-1 protein and regulatory machinery and how expanding knowledge of the function of individual HIV-1-coded proteins has provided valuable insights into understanding HIV transcriptional regulation in selected susceptible cell types. Historically, Tat has been the most studied primary transactivator protein, but emerging knowledge of HIV-1 transcriptional regulation in cells of the monocyte–macrophage lineage has more recently established that a number of the HIV-1 accessory proteins like Vpr may directly or indirectly regulate the transcriptional process. The viral proteins Nef and matrix play important roles in modulating the cellular activation pathways to facilitate viral replication. These observations highlight the cross talk between the HIV-1 transcriptional machinery and cellular activation pathways. The review also discusses the proposed transcriptional regulation mechanisms that intersect with the pathways regulated by microRNAs and how development of the knowledge of chromatin biology has enhanced our understanding of key protein–protein and protein–DNA interactions that form the HIV-1 transcriptome. Finally, we discuss the potential pharmacological approaches to target viral persistence and enhance effective transcription to purge the virus in cellular reservoirs, especially within the central nervous system, and the novel therapeutics that are currently in various stages of development to achieve a much superior prognosis for the HIV-1-infected population. PMID:22422068

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses

PubMed Central

Liang, Qin; Dexheimer, Thomas S; Zhang, Ping; Rosenthal, Andrew S; Villamil, Mark A; You, Changjun; Zhang, Qiuting; Chen, Junjun; Ott, Christine A; Sun, Hongmao; Luci, Diane K; Yuan, Bifeng; Simeonov, Anton; Jadhav, Ajit; Xiao, Hui; Wang, Yinsheng; Maloney, David J; Zhuang, Zhihao

2014-01-01

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs. PMID:24531842

UVA and UVB Irradiation Differentially Regulate microRNA Expression in Human Primary Keratinocytes

PubMed Central

Kraemer, Anne; Chen, I-Peng; Henning, Stefan; Faust, Alexandra; Volkmer, Beate; Atkinson, Michael J.; Moertl, Simone; Greinert, Ruediger

2013-01-01

MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis. PMID:24391759

Role of Vanadium in Cellular and Molecular Immunology: Association with Immune-Related Inflammation and Pharmacotoxicology Mechanisms

PubMed Central

Tsave, Olga; Petanidis, Savvas; Kioseoglou, Efrosini; Yavropoulou, Maria P.; Yovos, John G.; Anestakis, Doxakis; Tsepa, Androniki; Salifoglou, Athanasios

2016-01-01

Over the last decade, a diverse spectrum of vanadium compounds has arisen as anti-inflammatory therapeutic metallodrugs targeting various diseases. Recent studies have demonstrated that select well-defined vanadium species are involved in many immune-driven molecular mechanisms that regulate and influence immune responses. In addition, advances in cell immunotherapy have relied on the use of metallodrugs to create a “safe,” highly regulated, environment for optimal control of immune response. Emerging findings include optimal regulation of B/T cell signaling and expression of immune suppressive or anti-inflammatory cytokines, critical for immune cell effector functions. Furthermore, in-depth perusals have explored NF-κB and Toll-like receptor signaling mechanisms in order to enhance adaptive immune responses and promote recruitment or conversion of inflammatory cells to immunodeficient tissues. Consequently, well-defined vanadium metallodrugs, poised to access and resensitize the immune microenvironment, interact with various biomolecular targets, such as B cells, T cells, interleukin markers, and transcription factors, thereby influencing and affecting immune signaling. A synthetically formulated and structure-based (bio)chemical reactivity account of vanadoforms emerges as a plausible strategy for designing drugs characterized by selectivity and specificity, with respect to the cellular molecular targets intimately linked to immune responses, thereby giving rise to a challenging field linked to the development of immune system vanadodrugs. PMID:27190573

Methods in Molecular Biology: Germline Stem Cells | Center for Cancer Research

Cancer.gov

The protocols in Germline Stem Cells are intended to present selected genetic, molecular, and cellular techniques used in germline stem cell research. The book is divided into two parts. Part I covers germline stem cell identification and regulation in model organisms. Part II covers current techniques used in in vitro culture and applications of germline stem cells.

Rab GTPases and Membrane Trafficking in Neurodegeneration

PubMed Central

Kiral, Ferdi Ridvan; Kohrs, Friederike Elisabeth; Jin, Eugene Jennifer; Hiesinger, Peter Robin

2018-01-01

Defects in membrane trafficking are hallmarks of neurodegeneration. Rab GTPases are key regulators of membrane trafficking. Alterations of Rab GTPases, or the membrane compartments they regulate, are associated with virtually all neuronal activities in health and disease. The observation that many Rab GTPases are associated with neurodegeneration has proven a challenge in the quest for cause and effect. Neurodegeneration can be a direct consequence of a defect in membrane trafficking. Alternatively, changes in membrane trafficking may be secondary consequences or cellular responses. The secondary consequences and cellular responses, in turn, may protect, represent inconsequential correlates or function as drivers of pathology. Here, we attempt to disentangle the different roles of membrane trafficking in neurodegeneration by focusing on selected associations with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and selected neuropathies. We provide an overview of current knowledge on Rab GTPase functions in neurons and review the associations of Rab GTPases with neurodegeneration with respect to the following classifications: primary cause, secondary cause driving pathology or secondary correlate. This analysis is devised to aid the interpretation of frequently observed membrane trafficking defects in neurodegeneration and facilitate the identification of true causes of pathology. PMID:29689231

Biological Chemistry and Functionality of Protein Sulfenic Acids and Related Thiol Modifications

PubMed Central

Devarie-Baez, Nelmi O.; Silva Lopez, Elsa I.; Furdui, Cristina M.

2016-01-01

Selective modification of proteins at cysteine residues by reactive oxygen, nitrogen or sulfur species formed under physiological and pathological states is emerging as a critical regulator of protein activity impacting cellular function. This review focuses primarily on protein sulfenylation (-SOH), a metastable reversible modification connecting reduced cysteine thiols to many products of cysteine oxidation. An overview is first provided on the chemistry principles underlining synthesis, stability and reactivity of sulfenic acids in model compounds and proteins, followed by a brief description of analytical methods currently employed to characterize these oxidative species. The following chapters present a selection of redox-regulated proteins for which the -SOH formation was experimentally confirmed and linked to protein function. These chapters are organized based on the participation of these proteins in the regulation of signaling, metabolism and epigenetics. The last chapter discusses the therapeutic implications of altered redox microenvironment and protein oxidation in disease. PMID:26340608

Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain*

PubMed Central

Tokarski, John S.; Zupa-Fernandez, Adriana; Tredup, Jeffrey A.; Pike, Kristen; Chang, ChiehYing; Xie, Dianlin; Cheng, Lihong; Pedicord, Donna; Muckelbauer, Jodi; Johnson, Stephen R.; Wu, Sophie; Edavettal, Suzanne C.; Hong, Yang; Witmer, Mark R.; Elkin, Lisa L.; Blat, Yuval; Pitts, William J.; Weinstein, David S.; Burke, James R.

2015-01-01

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity. PMID:25762719

Structural Basis for the Potent and Selective Inhibition of Casein Kinase 1 Epsilon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Long, Alexander M.; Zhao, Huilin; Huang, Xin

2012-10-29

Casein kinase 1 epsilon (CK1ε) and its closest homologue CK1δ are key regulators of diverse cellular processes. We report two crystal structures of PF4800567, a potent and selective inhibitor of CK1ε, bound to the kinase domains of human CK1ε and CK1δ as well as one apo CK1ε crystal structure. These structures provide a molecular basis for the strong and specific inhibitor interactions with CK1ε and suggest clues for further development of CK1δ inhibitors.

Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

PubMed

Khajuria, Rajiv K; Munschauer, Mathias; Ulirsch, Jacob C; Fiorini, Claudia; Ludwig, Leif S; McFarland, Sean K; Abdulhay, Nour J; Specht, Harrison; Keshishian, Hasmik; Mani, D R; Jovanovic, Marko; Ellis, Steven R; Fulco, Charles P; Engreitz, Jesse M; Schütz, Sabina; Lian, John; Gripp, Karen W; Weinberg, Olga K; Pinkus, Geraldine S; Gehrke, Lee; Regev, Aviv; Lander, Eric S; Gazda, Hanna T; Lee, Winston Y; Panse, Vikram G; Carr, Steven A; Sankaran, Vijay G

2018-03-22

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

PubMed

Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

2009-08-07

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

Antioxidant enzymes as redox-based biomarkers: a brief review.

PubMed

Yang, Hee-Young; Lee, Tae-Hoon

2015-04-01

The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease.

Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

PubMed

Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J; Zimmer, Danna B; Honkanen, Richard E; Wadzinski, Brian E

2014-02-14

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

A unique hinge binder of extremely selective aminopyridine-based Mps1 (TTK) kinase inhibitors with cellular activity.

PubMed

Kusakabe, Ken-ichi; Ide, Nobuyuki; Daigo, Yataro; Itoh, Takeshi; Yamamoto, Takahiko; Kojima, Eiichi; Mitsuoka, Yasunori; Tadano, Genta; Tagashira, Sachie; Higashino, Kenichi; Okano, Yousuke; Sato, Yuji; Inoue, Makiko; Iguchi, Motofumi; Kanazawa, Takayuki; Ishioka, Yukichi; Dohi, Keiji; Kido, Yasuto; Sakamoto, Shingo; Ando, Shigeru; Maeda, Masahiro; Higaki, Masayo; Yoshizawa, Hidenori; Murai, Hitoshi; Nakamura, Yusuke

2015-05-01

Mps1, also known as TTK, is a dual-specificity kinase that regulates the spindle assembly check point. Increased expression levels of Mps1 are observed in cancer cells, and the expression levels correlate well with tumor grade. Such evidence points to selective inhibition of Mps1 as an attractive strategy for cancer therapeutics. Starting from an aminopyridine-based lead 3a that binds to a flipped-peptide conformation at the hinge region in Mps1, elaboration of the aminopyridine scaffold at the 2- and 6-positions led to the discovery of 19c that exhibited no significant inhibition for 287 kinases as well as improved cellular Mps1 and antiproliferative activities in A549 lung carcinoma cells (cellular Mps1 IC₅₀=5.3 nM, A549 IC₅₀=26 nM). A clear correlation between cellular Mps1 and antiproliferative IC₅₀ values indicated that the antiproliferative activity observed in A549 cells would be responsible for the cellular inhibition of Mps1. The X-ray structure of 19c in complex with Mps1 revealed that this compound retains the ability to bind to the peptide flip conformation. Finally, comparative analysis of the X-ray structures of 19c, a deamino analogue 33, and a known Mps1 inhibitor bound to Mps1 provided insights into the unique binding mode at the hinge region. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal.

PubMed

Putker, Marrit; O'Neill, John Stuart

2016-01-01

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping.

Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal

PubMed Central

Putker, Marrit; O’Neill, John Stuart

2016-01-01

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping. PMID:26810072

Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust.

PubMed

Go, Yoon Young; Park, Moo Kyun; Kwon, Jee Young; Seo, Young Rok; Chae, Sung-Won; Song, Jae-Jun

2015-12-01

The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media.

Myeloid differentiation architecture of leukocyte transcriptome dynamics in perceived social isolation

PubMed Central

Cole, Steven W.; Capitanio, John P.; Chun, Katie; Arevalo, Jesusa M. G.; Ma, Jeffrey; Cacioppo, John T.

2015-01-01

To define the cellular mechanisms of up-regulated inflammatory gene expression and down-regulated antiviral response in people experiencing perceived social isolation (loneliness), we conducted integrative analyses of leukocyte gene regulation in humans and rhesus macaques. Five longitudinal leukocyte transcriptome surveys in 141 older adults showed up-regulation of the sympathetic nervous system (SNS), monocyte population expansion, and up-regulation of the leukocyte conserved transcriptional response to adversity (CTRA). Mechanistic analyses in a macaque model of perceived social isolation confirmed CTRA activation and identified selective up-regulation of the CD14++/CD16− classical monocyte transcriptome, functional glucocorticoid desensitization, down-regulation of Type I and II interferons, and impaired response to infection by simian immunodeficiency virus (SIV). These analyses identify neuroendocrine-related alterations in myeloid cell population dynamics as a key mediator of CTRA transcriptome skewing, which may both propagate perceived social isolation and contribute to its associated health risks. PMID:26598672

Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit.

PubMed

Costales, Matthew G; Haga, Christopher L; Velagapudi, Sai Pradeep; Childs-Disney, Jessica L; Phinney, Donald G; Disney, Matthew D

2017-03-08

A hypoxic state is critical to the metastatic and invasive characteristics of cancer. Numerous pathways play critical roles in cancer maintenance, many of which include noncoding RNAs such as microRNA (miR)-210 that regulates hypoxia inducible factors (HIFs). Herein, we describe the identification of a small molecule named Targapremir-210 that binds to the Dicer site of the miR-210 hairpin precursor. This interaction inhibits production of the mature miRNA, derepresses glycerol-3-phosphate dehydrogenase 1-like enzyme (GPD1L), a hypoxia-associated protein negatively regulated by miR-210, decreases HIF-1α, and triggers apoptosis of triple negative breast cancer cells only under hypoxic conditions. Further, Targapremir-210 inhibits tumorigenesis in a mouse xenograft model of hypoxic triple negative breast cancer. Many factors govern molecular recognition of biological targets by small molecules. For protein, chemoproteomics and activity-based protein profiling are invaluable tools to study small molecule target engagement and selectivity in cells. Such approaches are lacking for RNA, leaving a void in the understanding of its druggability. We applied Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP) to study the cellular selectivity and the on- and off-targets of Targapremir-210. Targapremir-210 selectively recognizes the miR-210 precursor and can differentially recognize RNAs in cells that have the same target motif but have different expression levels, revealing this important feature for selectively drugging RNAs for the first time. These studies show that small molecules can be rapidly designed to selectively target RNAs and affect cellular responses to environmental conditions, resulting in favorable benefits against cancer. Further, they help define rules for identifying druggable targets in the transcriptome.

Phosphoinositide 3-Kinase p110β Regulates Integrin αIIbβ3 Avidity and the Cellular Transmission of Contractile Forces*

PubMed Central

Schoenwaelder, Simone M.; Ono, Akiko; Nesbitt, Warwick S.; Lim, Joanna; Jarman, Kate; Jackson, Shaun P.

2010-01-01

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin αIIbβ3, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin αIIbβ3 activation (affinity modulation) primarily occurs downstream of Gi-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin αIIbβ3 bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin αIIbβ3 activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin αIIbβ3 association with the contractile cytoskeleton. Analysis of integrin αIIbβ3 adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin αIIbβ3 bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin αIIbβ3 receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors. PMID:19940148

Emerging roles for neurosteroids in sexual behavior and function.

PubMed

King, Steven R

2008-01-01

Although gonadal and adrenal steroids heavily impact sexual function at the level of the brain, the nervous system also produces its own steroids de novo that may regulate sexual behavior and reproduction. Current evidence points to important roles for neurosteroids in sexual and gender-typical behaviors, control of ovulation, and behaviors that strongly influence sexual interest and motivation like aggression, anxiety and depression. At the cellular level, neurosteroids act through stimulating rapid changes in excitability and direct activation of membrane receptors in neurons. Thus, unlike peripheral steroids, neurosteroids can have immediate and specific effects on select neuronal pathways to regulate sexual function.

Genome-scale prediction of proteins with long intrinsically disordered regions.

PubMed

Peng, Zhenling; Mizianty, Marcin J; Kurgan, Lukasz

2014-01-01

Proteins with long disordered regions (LDRs), defined as having 30 or more consecutive disordered residues, are abundant in eukaryotes, and these regions are recognized as a distinct class of biologically functional domains. LDRs facilitate various cellular functions and are important for target selection in structural genomics. Motivated by the lack of methods that directly predict proteins with LDRs, we designed Super-fast predictor of proteins with Long Intrinsically DisordERed regions (SLIDER). SLIDER utilizes logistic regression that takes an empirically chosen set of numerical features, which consider selected physicochemical properties of amino acids, sequence complexity, and amino acid composition, as its inputs. Empirical tests show that SLIDER offers competitive predictive performance combined with low computational cost. It outperforms, by at least a modest margin, a comprehensive set of modern disorder predictors (that can indirectly predict LDRs) and is 16 times faster compared to the best currently available disorder predictor. Utilizing our time-efficient predictor, we characterized abundance and functional roles of proteins with LDRs over 110 eukaryotic proteomes. Similar to related studies, we found that eukaryotes have many (on average 30.3%) proteins with LDRs with majority of proteomes having between 25 and 40%, where higher abundance is characteristic to proteomes that have larger proteins. Our first-of-its-kind large-scale functional analysis shows that these proteins are enriched in a number of cellular functions and processes including certain binding events, regulation of catalytic activities, cellular component organization, biogenesis, biological regulation, and some metabolic and developmental processes. A webserver that implements SLIDER is available at http://biomine.ece.ualberta.ca/SLIDER/. Copyright © 2013 Wiley Periodicals, Inc.

Functional Properties of the Mitochondrial Carrier System.

PubMed

Taylor, Eric B

2017-09-01

The mitochondrial carrier system (MCS) transports small molecules between mitochondria and the cytoplasm. It is integral to the core mitochondrial function to regulate cellular chemistry by metabolism. The mammalian MCS comprises the transporters of the 53-member canonical SLC25A family and a lesser number of identified noncanonical transporters. The recent discovery and investigations of the mitochondrial pyruvate carrier (MPC) illustrate the diverse effects a single mitochondrial carrier may exert on cellular function. However, the transport selectivities of many carriers remain unknown, and most have not been functionally investigated in mammalian cells. The mechanisms coordinating their function as a unified system remain undefined. Increased accessibility to molecular genetic and metabolomic technologies now greatly enables investigation of the MCS. Continued investigation of the MCS may reveal how mitochondria encode complex regulatory information within chemical thermodynamic gradients. This understanding may enable precision modulation of cellular chemistry to counteract the dysmetabolism inherent in disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sleep and the Price of Plasticity: From Synaptic and Cellular Homeostasis to Memory Consolidation and Integration

PubMed Central

Tononi, Giulio; Cirelli, Chiara

2014-01-01

Summary Sleep is universal, tightly regulated, and its loss impairs cognition. But why does the brain need to disconnect from the environment for hours every day? The synaptic homeostasis hypothesis (SHY) proposes that sleep is the price the brain pays for plasticity. During a waking episode, learning statistical regularities about the current environment requires strengthening connections throughout the brain. This increases cellular needs for energy and supplies, decreases signal-to-noise ratios, and saturates learning. During sleep, spontaneous activity renormalizes net synaptic strength and restores cellular homeostasis. Activity-dependent down-selection of synapses can also explain the benefits of sleep on memory acquisition, consolidation, and integration. This happens through the off-line, comprehensive sampling of statistical regularities incorporated in neuronal circuits over a lifetime. This review considers the rationale and evidence for SHY and points to open issues related to sleep and plasticity. PMID:24411729

Sleep and the price of plasticity: from synaptic and cellular homeostasis to memory consolidation and integration.

PubMed

Tononi, Giulio; Cirelli, Chiara

2014-01-08

Sleep is universal, tightly regulated, and its loss impairs cognition. But why does the brain need to disconnect from the environment for hours every day? The synaptic homeostasis hypothesis (SHY) proposes that sleep is the price the brain pays for plasticity. During a waking episode, learning statistical regularities about the current environment requires strengthening connections throughout the brain. This increases cellular needs for energy and supplies, decreases signal-to-noise ratios, and saturates learning. During sleep, spontaneous activity renormalizes net synaptic strength and restores cellular homeostasis. Activity-dependent down-selection of synapses can also explain the benefits of sleep on memory acquisition, consolidation, and integration. This happens through the offline, comprehensive sampling of statistical regularities incorporated in neuronal circuits over a lifetime. This Perspective considers the rationale and evidence for SHY and points to open issues related to sleep and plasticity. Copyright © 2014 Elsevier Inc. All rights reserved.

The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

PubMed Central

Nakatsu, Fubito; Hase, Koji; Ohno, Hiroshi

2014-01-01

The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP)-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis). Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells. PMID:25387275

Estrogen Biology: New Insights into GPER Function and Clinical Opportunities

PubMed Central

Prossnitz, Eric R.; Barton, Matthias

2014-01-01

Estrogens play an important role in the regulation of normal physiology, aging and many disease states. Although the nuclear estrogen receptors have classically been described to function as ligand-activated transcription factors mediating genomic effects in hormonally regulated tissues, more recent studies reveal that estrogens also mediate rapid signaling events traditionally associated with G protein-coupled receptors. The G protein-coupled estrogen receptor GPER (formerly GPR30) has now become recognized as a major mediator of estrogen’s rapid cellular effects throughout the body. With the discovery of selective synthetic ligands for GPER, both agonists and antagonists, as well as the use of GPER knockout mice, significant advances have been made in our understanding of GPER function at the cellular, tissue and organismal levels. In many instances, the protective/beneficial effects of estrogen are mimicked by selective GPER agonism and are absent or reduced in GPER knockout mice, suggesting an essential or at least parallel role for GPER in the actions of estrogen. In this review, we will discuss recent advances and our current understanding of the role of GPER and certain drugs such as SERMs and SERDs in physiology and disease. We will also highlight novel opportunities for clinical development towards GPER-targeted therapeutics, for molecular imaging, as well as for theranostic approaches and personalized medicine. PMID:24530924

Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition

PubMed Central

Wu, Wen-I; Voegtli, Walter C.; Sturgis, Hillary L.; Dizon, Faith P.; Vigers, Guy P. A.; Brandhuber, Barbara J.

2010-01-01

AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones. PMID:20886116

Finding new pathway-specific regulators by clustering method using threshold standard deviation based on DNA chip data of Streptomyces coelicolor.

PubMed

Yang, Yung-Hun; Kim, Ji-Nu; Song, Eunjung; Kim, Eunjung; Oh, Min-Kyu; Kim, Byung-Gee

2008-09-01

In order to identify the regulators involved in antibiotic production or time-specific cellular events, the messenger ribonucleic acid (mRNA) expression data of the two gene clusters, actinorhodin (ACT) and undecylprodigiosin (RED) biosynthetic genes, were clustered with known mRNA expression data of regulators from S. coelicolor using a filtering method based on standard deviation and clustering analysis. The result identified five regulators including two well-known regulators namely, SCO3579 (WlbA) and SCO6722 (SsgD). Using overexpression and deletion of the regulator genes, we were able to identify two regulators, i.e., SCO0608 and SCO6808, playing roles as repressors in antibiotics production and sporulation. This approach can be easily applied to mapping out new regulators related to any interesting target gene clusters showing characteristic expression patterns. The result can also be used to provide insightful information on the selection rules among a large number of regulators.

Membrane fluidity controls redox-regulated cold stress responses in cyanobacteria.

PubMed

Maksimov, Eugene G; Mironov, Kirill S; Trofimova, Marina S; Nechaeva, Natalya L; Todorenko, Daria A; Klementiev, Konstantin E; Tsoraev, Georgy V; Tyutyaev, Eugene V; Zorina, Anna A; Feduraev, Pavel V; Allakhverdiev, Suleyman I; Paschenko, Vladimir Z; Los, Dmitry A

2017-09-01

Membrane fluidity is the important regulator of cellular responses to changing ambient temperature. Bacteria perceive cold by the transmembrane histidine kinases that sense changes in thickness of the cytoplasmic membrane due to its rigidification. In the cyanobacterium Synechocystis, about a half of cold-responsive genes is controlled by the light-dependent transmembrane histidine kinase Hik33, which also partially controls the responses to osmotic, salt, and oxidative stress. This implies the existence of some universal, but yet unknown signal that triggers adaptive gene expression in response to various stressors. Here we selectively probed the components of photosynthetic machinery and functionally characterized the thermodynamics of cyanobacterial photosynthetic membranes with genetically altered fluidity. We show that the rate of oxidation of the quinone pool (PQ), which interacts with both photosynthetic and respiratory electron transport chains, depends on membrane fluidity. Inhibitor-induced stimulation of redox changes in PQ triggers cold-induced gene expression. Thus, the fluidity-dependent changes in the redox state of PQ may universally trigger cellular responses to stressors that affect membrane properties.

Hantaviruses induce cell type- and viral species-specific host microRNA expression signatures

PubMed Central

Shin, Ok Sarah; Kumar, Mukesh; Yanagihara, Richard; Song, Jin-Won

2014-01-01

The mechanisms of hantavirus-induced modulation of host cellular immunity remain poorly understood. Recently, microRNAs (miRNAs) have emerged as a class of essential regulators of host immune response genes. To ascertain if differential host miRNA expression toward representative hantavirus species correlated with immune response genes, miRNA expression profiles were analyzed in human endothelial cells, macrophages and epithelial cells infected with pathogenic and nonpathogenic rodent- and shrew-borne hantaviruses. Distinct miRNA expression profiles were observed in a cell type- and viral species-specific pattern. A subset of miRNAs, including miR-151-5p and miR-1973, were differentially expressed between Hantaan virus and Prospect Hill virus. Pathway analyses confirmed that the targets of selected miRNAs were associated with inflammatory responses and innate immune receptor-mediated signaling pathways. Our data suggest that differential immune responses following hantavirus infection may be regulated in part by cellular miRNA through dysregulation of genes critical to the inflammatory process. PMID:24074584

Targeting protein neddylation: a novel therapeutic strategy for the treatment of cancer.

PubMed

Wang, Meng; Medeiros, Bruno C; Erba, Harry P; DeAngelo, Daniel J; Giles, Francis J; Swords, Ronan T

2011-03-01

The NEDD8 (neural precursor cell-expressed developmentally downregulated 8) conjugation pathway regulates the post-translational modification of oncogenic proteins. This pathway has important potential for cancer therapeutics. Several proteins vital in cancer biology are regulated by protein neddylation. These observations led to the development of a small molecule inhibitor that disrupts protein neddylation and leads to cancer cell death and important activity in early phase clinical trials. This review provides an extensive coverage of cellular protein homeostasis with particular emphasis on the NEDD8 conjugation pathway. Insights into a new investigational drug that specifically disrupts the NEDD8 pathway are discussed. The clinical data for this agent are also updated. Neddylation controls key cellular pathways found to be dysregulated in many cancers. Protein neddylation is a relatively under-explored pathway for pharmacologic inhibition in cancer. Selective disruption of this pathway has demonstrated clinical activity in patients with myeloid neoplasms and is worth exploring further in combination with other anti-leukemia agents.

Cellular manganese content is developmentally regulated in human dopaminergic neurons

NASA Astrophysics Data System (ADS)

Kumar, Kevin K.; Lowe, Edward W., Jr.; Aboud, Asad A.; Neely, M. Diana; Redha, Rey; Bauer, Joshua A.; Odak, Mihir; Weaver, C. David; Meiler, Jens; Aschner, Michael; Bowman, Aaron B.

2014-10-01

Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical `toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.

Gene expression changes in the coccolithophore Emiliania huxleyi after 500 generations of selection to ocean acidification

PubMed Central

Lohbeck, Kai T.; Riebesell, Ulf; Reusch, Thorsten B. H.

2014-01-01

Coccolithophores are unicellular marine algae that produce biogenic calcite scales and substantially contribute to marine primary production and carbon export to the deep ocean. Ongoing ocean acidification particularly impairs calcifying organisms, mostly resulting in decreased growth and calcification. Recent studies revealed that the immediate physiological response in the coccolithophore Emiliania huxleyi to ocean acidification may be partially compensated by evolutionary adaptation, yet the underlying molecular mechanisms are currently unknown. Here, we report on the expression levels of 10 candidate genes putatively relevant to pH regulation, carbon transport, calcification and photosynthesis in E. huxleyi populations short-term exposed to ocean acidification conditions after acclimation (physiological response) and after 500 generations of high CO2 adaptation (adaptive response). The physiological response revealed downregulation of candidate genes, well reflecting the concomitant decrease of growth and calcification. In the adaptive response, putative pH regulation and carbon transport genes were up-regulated, matching partial restoration of growth and calcification in high CO2-adapted populations. Adaptation to ocean acidification in E. huxleyi likely involved improved cellular pH regulation, presumably indirectly affecting calcification. Adaptive evolution may thus have the potential to partially restore cellular pH regulatory capacity and thereby mitigate adverse effects of ocean acidification. PMID:24827439

Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals

NASA Astrophysics Data System (ADS)

Paulmurugan, Ramasamy; Sekar, Narayana M.; Sekar, Thillai V.

2012-03-01

MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve therapeutic effect in living animals.

Alternative Polyadenylation Regulates CELF1/CUGBP1 Target Transcripts Following T Cell Activation

PubMed Central

Beisang, Daniel; Reilly, Cavan; Bohjanen, Paul R.

2014-01-01

Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3′ end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3′ untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3′ end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation PMID:25123787

HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Wen; Jones, Frank E., E-mail: fjones3@tulane.edu

2014-01-10

Highlights: •HER4/4ICD is an obligate coactivator for 37% of estrogen regulated genes. •HER4/4ICD coactivated genes selectively regulate estrogen stimulated proliferation. •Estrogen stimulated tumor cell migration occurs independent of HER4/4ICD. •Disrupting HER4/4ICD and ER coactivated gene expression may suppress breast cancer. -- Abstract: The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex ismore » unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of β-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, β-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the β-estradiol stimulated genes. Ingenuity Pathway Analysis of β-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.« less

Genes uniquely expressed in human growth plate chondrocytes uncover a distinct regulatory network.

PubMed

Li, Bing; Balasubramanian, Karthika; Krakow, Deborah; Cohn, Daniel H

2017-12-20

Chondrogenesis is the earliest stage of skeletal development and is a highly dynamic process, integrating the activities and functions of transcription factors, cell signaling molecules and extracellular matrix proteins. The molecular mechanisms underlying chondrogenesis have been extensively studied and multiple key regulators of this process have been identified. However, a genome-wide overview of the gene regulatory network in chondrogenesis has not been achieved. In this study, employing RNA sequencing, we identified 332 protein coding genes and 34 long non-coding RNA (lncRNA) genes that are highly selectively expressed in human fetal growth plate chondrocytes. Among the protein coding genes, 32 genes were associated with 62 distinct human skeletal disorders and 153 genes were associated with skeletal defects in knockout mice, confirming their essential roles in skeletal formation. These gene products formed a comprehensive physical interaction network and participated in multiple cellular processes regulating skeletal development. The data also revealed 34 transcription factors and 11,334 distal enhancers that were uniquely active in chondrocytes, functioning as transcriptional regulators for the cartilage-selective genes. Our findings revealed a complex gene regulatory network controlling skeletal development whereby transcription factors, enhancers and lncRNAs participate in chondrogenesis by transcriptional regulation of key genes. Additionally, the cartilage-selective genes represent candidate genes for unsolved human skeletal disorders.

TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

PubMed Central

Cáceres, Mónica; Ortiz, Liliana; Recabarren, Tatiana; Romero, Anibal; Colombo, Alicia; Leiva-Salcedo, Elías; Varela, Diego; Rivas, José; Silva, Ian; Morales, Diego; Campusano, Camilo; Almarza, Oscar; Simon, Felipe; Toledo, Hector; Park, Kang-Sik; Trimmer, James S.; Cerda, Oscar

2015-01-01

Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility. PMID:26110647

Antioxidant enzymes as redox-based biomarkers: a brief review

PubMed Central

Yang, Hee-Young; Lee, Tae-Hoon

2015-01-01

The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease. [BMB Reports 2015; 48(4): 200-208] PMID:25560698

Quantifying the entropic cost of cellular growth control

NASA Astrophysics Data System (ADS)

De Martino, Daniele; Capuani, Fabrizio; De Martino, Andrea

2017-07-01

Viewing the ways a living cell can organize its metabolism as the phase space of a physical system, regulation can be seen as the ability to reduce the entropy of that space by selecting specific cellular configurations that are, in some sense, optimal. Here we quantify the amount of regulation required to control a cell's growth rate by a maximum-entropy approach to the space of underlying metabolic phenotypes, where a configuration corresponds to a metabolic flux pattern as described by genome-scale models. We link the mean growth rate achieved by a population of cells to the minimal amount of metabolic regulation needed to achieve it through a phase diagram that highlights how growth suppression can be as costly (in regulatory terms) as growth enhancement. Moreover, we provide an interpretation of the inverse temperature β controlling maximum-entropy distributions based on the underlying growth dynamics. Specifically, we show that the asymptotic value of β for a cell population can be expected to depend on (i) the carrying capacity of the environment, (ii) the initial size of the colony, and (iii) the probability distribution from which the inoculum was sampled. Results obtained for E. coli and human cells are found to be remarkably consistent with empirical evidence.

Autophagy Driven by a Master Regulator of Hematopoiesis

PubMed Central

Kang, Yoon-A; Sanalkumar, Rajendran; O'Geen, Henriette; Linnemann, Amelia K.; Chang, Chan-Jung; Bouhassira, Eric E.; Farnham, Peggy J.; Keles, Sunduz

2012-01-01

Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy. PMID:22025678

Calcium movements and the cellular basis of gravitropism

NASA Astrophysics Data System (ADS)

Roux, S. J.; Biro, R. L.; Hale, C. C.

An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.

Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

PubMed

Leininger, Elizabeth C; Kelley, Darcy B

2015-01-01

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Muscle carnitine availability plays a central role in regulating fuel metabolism in the rodent.

PubMed

Porter, Craig; Constantin-Teodosiu, Dumitru; Constantin, Despina; Leighton, Brendan; Poucher, Simon M; Greenhaff, Paul L

2017-09-01

Meldonium inhibits endogenous carnitine synthesis and tissue uptake, and accelerates urinary carnitine excretion, although the impact of meldonium-mediated muscle carnitine depletion on whole-body fuel selection, and muscle fuel metabolism and its molecular regulation is under-investigated. Ten days of oral meldonium administration did not impact on food or fluid intake, physical activity levels or body weight gain in the rat, whereas it depleted muscle carnitine content (all moieties), increased whole-body carbohydrate oxidation and muscle and liver glycogen utilization, and reduced whole-body fat oxidation. Meldonium reduced carnitine transporter protein expression across muscles of different contractile and metabolic phenotypes. A TaqMan PCR low-density array card approach revealed the abundance of 189 mRNAs regulating fuel selection was altered in soleus muscle by meldonium, highlighting the modulation of discrete cellular functions and metabolic pathways. These novel findings strongly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo. The body carnitine pool is primarily confined to skeletal muscle, where it regulates carbohydrate (CHO) and fat usage. Meldonium (3-(2,2,2-trimethylhydrazinium)-propionate) inhibits carnitine synthesis and tissue uptake, although the impact of carnitine depletion on whole-body fuel selection, muscle fuel metabolism and its molecular regulation is under-investigated. Male lean Zucker rats received water (control, n = 8) or meldonium-supplemented water (meldonium, n = 8) for 10 days [1.6 g kg -1 body mass (BM) day -1 days 1-2, 0.8 g kg -1  BM day -1 thereafter]. From days 7-10, animals were housed in indirect calorimetry chambers after which soleus muscle and liver were harvested. Food and fluid intake, weight gain and physical activity levels were similar between groups from days 7 to 10. Compared to control, meldonium depleted muscle total carnitine (P < 0.001) and all carnitine esters. Furthermore, whole-body fat oxidation was less (P < 0.001) and CHO oxidation was greater (P < 0.05) compared to the control, whereas soleus and liver glycogen contents were less (P < 0.01 and P < 0.01, respectively). In a second study, male Wistar rats received water (n = 8) or meldonium-supplemented water (n = 8) as above, and kidney, heart and extensor digitorum longus muscle (EDL) and soleus muscles were collected. Compared to control, meldonium depleted total carnitine content (all P < 0.001), reduced carnitine transporter protein and glycogen content, and increased pyruvate dehydrogenase kinase 4 mRNA abundance in the heart, EDL and soleus. In total, 189 mRNAs regulating fuel selection were differentially expressed in soleus in meldonium vs. control, and a number of cellular functions and pathways strongly associated with carnitine depletion were identified. Collectively, these data firmly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo. © 2017 The University of Nottingham. The Journal of Physiology © 2017 The Physiological Society.

Differential regulation of striatal motor behavior and related cellular responses by dopamine D2L and D2S isoforms.

PubMed

Radl, Daniela; Chiacchiaretta, Martina; Lewis, Robert G; Brami-Cherrier, Karen; Arcuri, Ludovico; Borrelli, Emiliana

2018-01-02

The dopamine D2 receptor (D2R) is a major component of the dopamine system. D2R-mediated signaling in dopamine neurons is involved in the presynaptic regulation of dopamine levels. Postsynaptically, i.e., in striatal neurons, D2R signaling controls complex functions such as motor activity through regulation of cell firing and heterologous neurotransmitter release. The presence of two isoforms, D2L and D2S, which are generated by a mechanism of alternative splicing of the Drd2 gene, raises the question of whether both isoforms may equally control presynaptic and postsynaptic events. Here, we addressed this question by comparing behavioral and cellular responses of mice with the selective ablation of either D2L or D2S isoform. We establish that the presence of either D2L or D2S can support postsynaptic functions related to the control of motor activity in basal conditions. On the contrary, absence of D2S but not D2L prevents the inhibition of tyrosine hydroxylase phosphorylation and, thereby, of dopamine synthesis, supporting a major presynaptic role for D2S. Interestingly, boosting dopamine signaling in the striatum by acute cocaine administration reveals that absence of D2L, but not of D2S, strongly impairs the motor and cellular response to the drug, in a manner similar to the ablation of both isoforms. These results suggest that when the dopamine system is challenged, D2L signaling is required for the control of striatal circuits regulating motor activity. Thus, our findings show that D2L and D2S share similar functions in basal conditions but not in response to stimulation of the dopamine system.

Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

PubMed Central

Mattoscio, Domenico; Casadio, Chiara; Miccolo, Claudia; Maffini, Fausto; Raimondi, Andrea; Tacchetti, Carlo; Gheit, Tarik; Tagliabue, Marta; Galimberti, Viviana E.; De Lorenzi, Francesca; Chiesa, Fausto; Ansarin, Mohssen; Tommasino, Massimo

2017-01-01

UBC9, the sole E2-conjugating enzyme required for SUMOylation, is a key regulator of essential cellular functions and, as such, is frequently altered in cancers. Along these lines, we recently reported that its expression gradually increases during early stages of human papillomavirus (HPV)-mediated cervical lesions transformation. However, a better understanding of how UBC9 is exploited by transforming viral oncoproteins is still needed. In the present study, we show that in human samples HPV drives UBC9 up-regulation also in very early steps of head and neck tumorigenesis, pointing to the important role for UBC9 in the HPV-mediated carcinogenic program. Moreover, using HPV-infected pre-cancerous tissues and primary human keratinocytes as the natural host of the virus, we investigate the pathological meaning and the cellular mechanisms responsible for UBC9 de-regulation in an oncoviral context. Our results show that UBC9 overexpression is promoted by transforming viral proteins to increase host cells’ resistance to apoptosis. In addition, ultrastuctural, pharmacological and genetic approaches crucially unveil that UBC9 is physiologically targeted by autophagy in human cells. However, the presence of HPV E6/E7 oncoproteins negatively impacts the autophagic process through selective inhibition of autophagosome-lysosome fusion, finally leading to p53 dependent UBC9 accumulation during viral-induced cellular transformation. Therefore, our study elucidates how UBC9 is manipulated by HPV oncoproteins, details the physiological mechanism by which UBC9 is degraded in cells, and identifies how HPV E6/E7 impact on autophagy. These findings point to UBC9 and autophagy as novel hallmarks of HPV oncogenesis, and open innovative avenues towards the treatment of HPV-related malignancies. PMID:28253371

MINCR is a MYC-induced lncRNA able to modulate MYC's transcriptional network in Burkitt lymphoma cells.

PubMed

Doose, Gero; Haake, Andrea; Bernhart, Stephan H; López, Cristina; Duggimpudi, Sujitha; Wojciech, Franziska; Bergmann, Anke K; Borkhardt, Arndt; Burkhardt, Birgit; Claviez, Alexander; Dimitrova, Lora; Haas, Siegfried; Hoell, Jessica I; Hummel, Michael; Karsch, Dennis; Klapper, Wolfram; Kleo, Karsten; Kretzmer, Helene; Kreuz, Markus; Küppers, Ralf; Lawerenz, Chris; Lenze, Dido; Loeffler, Markus; Mantovani-Löffler, Luisa; Möller, Peter; Ott, German; Richter, Julia; Rohde, Marius; Rosenstiel, Philip; Rosenwald, Andreas; Schilhabel, Markus; Schneider, Markus; Scholz, Ingrid; Stilgenbauer, Stephan; Stunnenberg, Hendrik G; Szczepanowski, Monika; Trümper, Lorenz; Weniger, Marc A; Hoffmann, Steve; Siebert, Reiner; Iaccarino, Ingram

2015-09-22

Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

BAD: undertaker by night, candyman by day.

PubMed

Danial, N N

2008-12-01

The BH3-only pro-apoptotic proteins are upstream sensors of cellular damage that selectively respond to specific, proximal death and survival signals. Genetic models and biochemical studies indicate that these molecules are latent killers until activated through transcriptional or post-translational mechanisms in a tissue-restricted and signal-specific manner. The large number of BH3-only proteins, their unique subcellular localization, protein-interaction network and diverse modes of activation suggest specialization of their damage-sensing function, ensuring that the core apoptotic machinery is poised to receive input from a wide range of cellular stress signals. The apoptotic response initiated by the activation of BH3-only proteins ultimately culminates in allosteric activation of pro-apoptotic BAX and BAK, the gateway proteins to the mitochondrial pathway of apoptosis. From activation of BH3-only proteins to oligomerization of BAX and BAK and mitochondrial outer membrane permeabilization, an intricate network of interactions between the pro- and anti-apoptotic members of the BCL-2 family orchestrates the decision to undergo apoptosis. Beyond regulation of apoptosis, multiple BCL-2 proteins have recently emerged as active components of select homeostatic pathways carrying other cellular functions. This review focuses on BAD, which was the first BH3-only protein linked to proximal survival signals through phosphorylation by survival kinases. In addition to findings that delineated the physiological role of BAD in apoptosis and its dynamic regulation by phosphorylation, studies pointing to new roles for this protein in other physiological pathways, such as glucose metabolism, are highlighted. By executing its 'day' and 'night' jobs in metabolism and apoptosis, respectively, BAD helps coordinate mitochondrial fuel metabolism and the apoptotic machinery.

Regulation of Sodium Transport in the Inner Ear

PubMed Central

Kim, Sung Huhn; Marcus, Daniel C.

2011-01-01

Na+ concentrations in endolymph must be controlled to maintain hair cell function since the transduction channels of hair cells are cation-permeable, but not K+-selective. Flooding or fluctuations of the hair cell cytosol with Na+ would be expected to lead to cellular dysfunction, hearing loss and vertigo. This review briefly describes cellular mechanisms known to be responsible for Na+homeostasis in each compartment of the inner ear, including the cochlea, saccule, semicircular canals and endolymphatic sac. The influx of Na+into endolymph of each of the organs is likely via passive diffusion, but these pathways have not yet been identified or characterized. Na+ absorption is controlled by gate -keeper channels in the apical (endolymphatic) membrane of the transporting cells. Highly Na+-selective epithelial sodium channels (ENaC) control absorption by Reissner’s membrane, saccular extramacular epithelium, semicircular canal duct epithelium and endolymphatic sac. ENaC activity is controlled by a number of signal pathways, but most notably by genomic regulation of channel numbers in the membrane via glucocorticoid signaling. Nonselective cation channels in the apical membrane of outer sulcus epithelial cells and vestibular transitional cells mediate Na+ and parasensory K+ absorption. The K+-mediated transduction current in hair cells is also accompanied by a Na+ flux since the transduction channels are nonselective cation channels. Cation absorption by all of these cells is regulated by extracellular ATP via apical nonselective cation channels (P2X receptors). The heterogeneous population of epithelial cells in the endolymphatic sac is thought to have multiple absorptive pathways for Na+ with regulatory pathways that include glucocorticoids and purinergic agonists. PMID:21620939

Distinct ontogenic and regional expressions of newly identified Cajal-Retzius cell-specific genes during neocorticogenesis.

PubMed

Yamazaki, Hiroshi; Sekiguchi, Mariko; Takamatsu, Masako; Tanabe, Yasuto; Nakanishi, Shigetada

2004-10-05

Cajal-Retzius (CR) cells are early-generated transient neurons and are important in the regulation of cortical neuronal migration and cortical laminar formation. Molecular entities characterizing the CR cell identity, however, remain largely elusive. We purified mouse cortical CR cells expressing GFP to homogeneity by fluorescence-activated cell sorting and examined a genome-wide expression profile of cortical CR cells at embryonic and postnatal periods. We identified 49 genes that exceeded hybridization signals by >10-fold in CR cells compared with non-CR cells at embryonic day 13.5, postnatal day 2, or both. Among these CR cell-specific genes, 25 genes, including the CR cell marker genes such as the reelin and calretinin genes, are selectively and highly expressed in both embryonic and postnatal CR cells. These genes, which encode generic properties of CR cell specificity, are eminently characterized as modulatory composites of voltage-dependent calcium channels and sets of functionally related cellular components involved in cell migration, adhesion, and neurite extension. Five genes are highly expressed in CR cells at the early embryonic period and are rapidly down-regulated thereafter. Furthermore, some of these genes have been shown to mark two distinctly different focal regions corresponding to the CR cell origins. At the late prenatal and postnatal periods, 19 genes are selectively up-regulated in CR cells. These genes include functional molecules implicated in synaptic transmission and modulation. CR cells thus strikingly change their cellular phenotypes during cortical development and play a pivotal role in both corticogenesis and cortical circuit maturation.

Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells.

PubMed

Streicher, Carmen; Heyny, Alexandra; Andrukhova, Olena; Haigl, Barbara; Slavic, Svetlana; Schüler, Christiane; Kollmann, Karoline; Kantner, Ingrid; Sexl, Veronika; Kleiter, Miriam; Hofbauer, Lorenz C; Kostenuik, Paul J; Erben, Reinhold G

2017-07-25

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

Development of Novel Therapeutics Targeting Isocitrate Dehydrogenase Mutations in Cancer.

PubMed

Sharma, Horrick

2018-05-17

Isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that catalyze the conversion of isocitrate to α-ketoglutarate (αKG). IDH 1 and IDH2 regulate several cellular processes, including oxidative respiration, glutamine metabolism, lipogenesis, and cellular defense against oxidative damage. Mutations in IDH1 and IDH2 have recently been observed in multiple tumor types, including gliomas, acute myeloid leukemia, myelodysplastic syndromes, and chondrosarcoma. IDH1 and IDH2 mutations involve a gain in neomorphic activity that catalyze αKG conversion to (R)-2-hydroxyglutarate ((R)-2HG). IDH mutation-mediated accumulation of (R)-2HG result in epigenetic dysregulation, altered gene expression, and a block in cellular differentiation. Targeting mutant IDH by development of small molecule inhibitors is a rapidly emerging therapeutic approach as evidenced by the recent approval of the first selective mutant IDH2 inhibitor AG-221 (Enasidenib) for the treatment of IDH2-mutated AML. This review will focus on mutant isocitrate dehydrogenase as a therapeutic drug target and provides an update on selective and pan-mutant IDH 1/2 inhibitors in clinical trials and other mutant IDH inhibitors that are under development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-07-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

PubMed Central

Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

2015-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations ( healthy young UK men and Scandinavian diabetic patients ) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold ( P  < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P  < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

Microarray analysis of gene expression alteration in human middle ear epithelial cells induced by micro particle.

PubMed

Song, Jae-Jun; Kwon, Jee Young; Park, Moo Kyun; Seo, Young Rok

2013-10-01

The primary aim of this study is to reveal the effect of particulate matter (PM) on the human middle ear epithelial cell (HMEEC). The HMEEC was treated with PM (300 μg/ml) for 24 h. Total RNA was extracted and used for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed by using Pathway Studio 9.0 software. For selected genes, the changes in gene expression were confirmed by real-time PCR. A total of 611 genes were regulated by PM. Among them, 366 genes were up-regulated, whereas 245 genes were down-regulated. Up-regulated genes were mainly involved in cellular processes, including reactive oxygen species generation, cell proliferation, apoptosis, cell differentiation, inflammatory response and immune response. Down-regulated genes affected several cellular processes, including cell differentiation, cell cycle, proliferation, apoptosis and cell migration. A total of 21 genes were discovered as crucial components in potential signaling networks containing 2-fold up regulated genes. Four genes, VEGFA, IL1B, CSF2 and HMOX1 were revealed as key mediator genes among the up-regulated genes. A total of 25 genes were revealed as key modulators in the signaling pathway associated with 2-fold down regulated genes. Four genes, including IGF1R, TIMP1, IL6 and FN1, were identified as the main modulator genes. We identified the differentially expressed genes in PM-treated HMEEC, whose expression profile may provide a useful clue for the understanding of environmental pathophysiology of otitis media. Our work indicates that air pollution, like PM, plays an important role in the pathogenesis of otitis media. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Estrogen biology: new insights into GPER function and clinical opportunities.

PubMed

Prossnitz, Eric R; Barton, Matthias

2014-05-25

Estrogens play an important role in the regulation of normal physiology, aging and many disease states. Although the nuclear estrogen receptors have classically been described to function as ligand-activated transcription factors mediating genomic effects in hormonally regulated tissues, more recent studies reveal that estrogens also mediate rapid signaling events traditionally associated with G protein-coupled receptors. The G protein-coupled estrogen receptor GPER (formerly GPR30) has now become recognized as a major mediator of estrogen's rapid cellular effects throughout the body. With the discovery of selective synthetic ligands for GPER, both agonists and antagonists, as well as the use of GPER knockout mice, significant advances have been made in our understanding of GPER function at the cellular, tissue and organismal levels. In many instances, the protective/beneficial effects of estrogen are mimicked by selective GPER agonism and are absent or reduced in GPER knockout mice, suggesting an essential or at least parallel role for GPER in the actions of estrogen. In this review, we will discuss recent advances and our current understanding of the role of GPER and the activity of clinically used drugs, such as SERMs and SERDs, in physiology and disease. We will also highlight novel opportunities for clinical development towards GPER-targeted therapeutics, for molecular imaging, as well as for theranostic approaches and personalized medicine. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks

NASA Astrophysics Data System (ADS)

White, Forest M.; Wolf-Yadlin, Alejandro

2016-06-01

Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs)

PubMed Central

Yang, Kai; Jackson, Michael F.; MacDonald, John F.

2014-01-01

G Protein Coupled Receptors (GPCRs) are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs), which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS) and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR) stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity. PMID:24562329

Genetics and epigenetics of aging and longevity

PubMed Central

Moskalev, Alexey A; Aliper, Alexander M; Smit-McBride, Zeljka; Buzdin, Anton; Zhavoronkov, Alex

2014-01-01

Evolutionary theories of aging predict the existence of certain genes that provide selective advantage early in life with adverse effect on lifespan later in life (antagonistic pleiotropy theory) or longevity insurance genes (disposable soma theory). Indeed, the study of human and animal genetics is gradually identifying new genes that increase lifespan when overexpressed or mutated: gerontogenes. Furthermore, genetic and epigenetic mechanisms are being identified that have a positive effect on longevity. The gerontogenes are classified as lifespan regulators, mediators, effectors, housekeeping genes, genes involved in mitochondrial function, and genes regulating cellular senescence and apoptosis. In this review we demonstrate that the majority of the genes as well as genetic and epigenetic mechanisms that are involved in regulation of longevity are highly interconnected and related to stress response. PMID:24603410

A large shRNA library approach identifies lncRNA Ntep as an essential regulator of cell proliferation

PubMed Central

Beermann, Julia; Kirste, Dominique; Iwanov, Katharina; Lu, Dongchao; Kleemiß, Felix; Kumarswamy, Regalla; Schimmel, Katharina; Bär, Christian; Thum, Thomas

2018-01-01

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach. PMID:29099486

Endoplasmic Reticulum Ca2+ Handling in Excitable Cells in Health and Disease

PubMed Central

Mattson, Mark P.

2011-01-01

The endoplasmic reticulum (ER) is a morphologically and functionally diverse organelle capable of integrating multiple extracellular and internal signals and generating adaptive cellular responses. It plays fundamental roles in protein synthesis and folding and in cellular responses to metabolic and proteotoxic stress. In addition, the ER stores and releases Ca2+ in sophisticated scenarios that regulate a range of processes in excitable cells throughout the body, including muscle contraction and relaxation, endocrine regulation of metabolism, learning and memory, and cell death. One or more Ca2+ ATPases and two types of ER membrane Ca2+ channels (inositol trisphosphate and ryanodine receptors) are the major proteins involved in ER Ca2+ uptake and release, respectively. There are also direct and indirect interactions of ER Ca2+ stores with plasma membrane and mitochondrial Ca2+-regulating systems. Pharmacological agents that selectively modify ER Ca2+ release or uptake have enabled studies that revealed many different physiological roles for ER Ca2+ signaling. Several inherited diseases are caused by mutations in ER Ca2+-regulating proteins, and perturbed ER Ca2+ homeostasis is implicated in a range of acquired disorders. Preclinical investigations suggest a therapeutic potential for use of agents that target ER Ca2+ handling systems of excitable cells in disorders ranging from cardiac arrhythmias and skeletal muscle myopathies to Alzheimer disease. PMID:21737534

Gene expression changes in the coccolithophore Emiliania huxleyi after 500 generations of selection to ocean acidification.

PubMed

Lohbeck, Kai T; Riebesell, Ulf; Reusch, Thorsten B H

2014-07-07

Coccolithophores are unicellular marine algae that produce biogenic calcite scales and substantially contribute to marine primary production and carbon export to the deep ocean. Ongoing ocean acidification particularly impairs calcifying organisms, mostly resulting in decreased growth and calcification. Recent studies revealed that the immediate physiological response in the coccolithophore Emiliania huxleyi to ocean acidification may be partially compensated by evolutionary adaptation, yet the underlying molecular mechanisms are currently unknown. Here, we report on the expression levels of 10 candidate genes putatively relevant to pH regulation, carbon transport, calcification and photosynthesis in E. huxleyi populations short-term exposed to ocean acidification conditions after acclimation (physiological response) and after 500 generations of high CO2 adaptation (adaptive response). The physiological response revealed downregulation of candidate genes, well reflecting the concomitant decrease of growth and calcification. In the adaptive response, putative pH regulation and carbon transport genes were up-regulated, matching partial restoration of growth and calcification in high CO2-adapted populations. Adaptation to ocean acidification in E. huxleyi likely involved improved cellular pH regulation, presumably indirectly affecting calcification. Adaptive evolution may thus have the potential to partially restore cellular pH regulatory capacity and thereby mitigate adverse effects of ocean acidification. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Redox Regulation of Protein Kinases

PubMed Central

Truong, Thu H.; Carroll, Kate S.

2015-01-01

Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

Prostate cancer-associated gene expression alterations determined from needle biopsies.

PubMed

Qian, David Z; Huang, Chung-Ying; O'Brien, Catherine A; Coleman, Ilsa M; Garzotto, Mark; True, Lawrence D; Higano, Celestia S; Vessella, Robert; Lange, Paul H; Nelson, Peter S; Beer, Tomasz M

2009-05-01

To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays composed of 6,200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative reverse transcription-PCR. Comparative analyses identified 954 transcript alterations associated with cancer (q < 0.01%), including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy use, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of androgen receptor expression changes was noted. In exploratory analyses, androgen receptor down-regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.

Prostate Cancer-Associated Gene Expression Alterations Determined from Needle Biopsies

PubMed Central

Qian, David Z.; Huang, Chung-Ying; O'Brien, Catherine A.; Coleman, Ilsa M.; Garzotto, Mark; True, Lawrence D.; Higano, Celestia S.; Vessella, Robert; Lange, Paul H.; Nelson, Peter S.; Beer, Tomasz M.

2010-01-01

Purpose To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. Experimental Design Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays comprised of 6200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative RT-PCR. Results Comparative analyses identified 954 transcript alterations associated with cancer (q value <0.01%) including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy utilization, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of AR expression changes was noted. In exploratory analyses, AR down regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. Conclusions Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation. PMID:19366833

Hacking the Cell: Network Intrusion and Exploitation by Adenovirus E1A.

PubMed

King, Cason R; Zhang, Ali; Tessier, Tanner M; Gameiro, Steven F; Mymryk, Joe S

2018-05-01

As obligate intracellular parasites, viruses are dependent on their infected hosts for survival. Consequently, viruses are under enormous selective pressure to utilize available cellular components and processes to their own advantage. As most, if not all, cellular activities are regulated at some level via protein interactions, host protein interaction networks are particularly vulnerable to viral exploitation. Indeed, viral proteins frequently target highly connected "hub" proteins to "hack" the cellular network, defining the molecular basis for viral control over the host. This widespread and successful strategy of network intrusion and exploitation has evolved convergently among numerous genetically distinct viruses as a result of the endless evolutionary arms race between pathogens and hosts. Here we examine the means by which a particularly well-connected viral hub protein, human adenovirus E1A, compromises and exploits the vulnerabilities of eukaryotic protein interaction networks. Importantly, these interactions identify critical regulatory hubs in the human proteome and help define the molecular basis of their function. Copyright © 2018 King et al.

Hacking the Cell: Network Intrusion and Exploitation by Adenovirus E1A

PubMed Central

King, Cason R.; Zhang, Ali; Tessier, Tanner M.; Gameiro, Steven F.

2018-01-01

ABSTRACT As obligate intracellular parasites, viruses are dependent on their infected hosts for survival. Consequently, viruses are under enormous selective pressure to utilize available cellular components and processes to their own advantage. As most, if not all, cellular activities are regulated at some level via protein interactions, host protein interaction networks are particularly vulnerable to viral exploitation. Indeed, viral proteins frequently target highly connected “hub” proteins to “hack” the cellular network, defining the molecular basis for viral control over the host. This widespread and successful strategy of network intrusion and exploitation has evolved convergently among numerous genetically distinct viruses as a result of the endless evolutionary arms race between pathogens and hosts. Here we examine the means by which a particularly well-connected viral hub protein, human adenovirus E1A, compromises and exploits the vulnerabilities of eukaryotic protein interaction networks. Importantly, these interactions identify critical regulatory hubs in the human proteome and help define the molecular basis of their function. PMID:29717008

Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle.

PubMed

Bayguinov, O; Dwyer, L; Kim, H; Marklew, A; Sanders, K M; Koh, S D

2011-06-01

The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents. The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

PubMed

Zhou, Nan; Yao, Yu; Ye, Hongxing; Zhu, Wei; Chen, Liang; Mao, Ying

2016-04-15

Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway. © 2015 UICC.

Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy.

PubMed

Izumi, Hiroto; Torigoe, Takayuki; Ishiguchi, Hiroshi; Uramoto, Hidetaka; Yoshida, Yoichiro; Tanabe, Mizuho; Ise, Tomoko; Murakami, Tadashi; Yoshida, Takeshi; Nomoto, Minoru; Kohno, Kimitoshi

2003-12-01

One of the major obstacles to the successful treatment of cancer is the complex biology of solid tumour development. Although regulation of intracellular pH has been shown to be critically important for many cellular functions, pH regulation has not been fully investigated in the field of cancer. It has, however, been shown that cellular pH is crucial for biological functions such as cell proliferation, invasion and metastasis, drug resistance and apoptosis. Hypoxic conditions are often observed during the development of solid tumours and lead to intracellular and extracellular acidosis. Cellular acidosis has been shown to be a trigger in the early phase of apoptosis and leads to activation of endonucleases inducing DNA fragmentation. To avoid intracellular acidification under such conditions, pH regulators are thought to be up-regulated in tumour cells. Four major types of pH regulator have been identified: the proton pump, the sodium-proton exchanger family (NHE), the bicarbonate transporter family (BCT) and the monocarboxylate transporter family (MCT). Here, we describe the structure and function of pH regulators expressed in tumour tissue. Understanding pH regulation in tumour cells may provide new ways of inducing tumour-specific apoptosis, thus aiding cancer chemotherapy.

Genomic signal processing: from matrix algebra to genetic networks.

PubMed

Alter, Orly

2007-01-01

DNA microarrays make it possible, for the first time, to record the complete genomic signals that guide the progression of cellular processes. Future discovery in biology and medicine will come from the mathematical modeling of these data, which hold the key to fundamental understanding of life on the molecular level, as well as answers to questions regarding diagnosis, treatment, and drug development. This chapter reviews the first data-driven models that were created from these genome-scale data, through adaptations and generalizations of mathematical frameworks from matrix algebra that have proven successful in describing the physical world, in such diverse areas as mechanics and perception: the singular value decomposition model, the generalized singular value decomposition model comparative model, and the pseudoinverse projection integrative model. These models provide mathematical descriptions of the genetic networks that generate and sense the measured data, where the mathematical variables and operations represent biological reality. The variables, patterns uncovered in the data, correlate with activities of cellular elements such as regulators or transcription factors that drive the measured signals and cellular states where these elements are active. The operations, such as data reconstruction, rotation, and classification in subspaces of selected patterns, simulate experimental observation of only the cellular programs that these patterns represent. These models are illustrated in the analyses of RNA expression data from yeast and human during their cell cycle programs and DNA-binding data from yeast cell cycle transcription factors and replication initiation proteins. Two alternative pictures of RNA expression oscillations during the cell cycle that emerge from these analyses, which parallel well-known designs of physical oscillators, convey the capacity of the models to elucidate the design principles of cellular systems, as well as guide the design of synthetic ones. In these analyses, the power of the models to predict previously unknown biological principles is demonstrated with a prediction of a novel mechanism of regulation that correlates DNA replication initiation with cell cycle-regulated RNA transcription in yeast. These models may become the foundation of a future in which biological systems are modeled as physical systems are today.

Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Human immunodeficiency virus (HIV) type 1 Vpr induces differential regulation of T cell costimulatory molecules: Direct effect of Vpr on T cell activation and immune function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkatachari, Narasimhan J.; Majumder, Biswanath; Ayyavoo, Velpandi

2007-02-20

Human immunodeficiency virus type 1 (HIV-1) viral proteins disrupt the normal host cellular immune pathways thus exploiting the cellular machinery for replication, survival and to escape host immune attack. Here we evaluated the direct effects of HIV-1 Vpr-mediated immune modulation of infected T cells. Vpr specifically downregulated the expression of CD28 and increased the expression of CTLA-4, whereas no significant difference in the expression of CD25 and HLA-DR was observed. Interferon gamma (IFN-{gamma}) production in T cells was evaluated as a measure of the downstream effector functions. Results indicate that Vpr significantly inhibited IFN-{gamma} production and this may, in part,more » due to Vpr's ability to inhibit the nuclear translocation of NF-{kappa}B, and its transcriptional regulation. Together these results support that HIV-1 Vpr selectively dysregulates the immune functions at multiple levels and exerts its inhibitory effects in the presence of other viral proteins.« less

Kv10.1 potassium channel: from the brain to the tumors.

PubMed

Cázares-Ordoñez, V; Pardo, L A

2017-10-01

The KCNH1 gene encodes the Kv10.1 (Eag1) ion channel, a member of the EAG (ether-à-go-go) family of voltage-gated potassium channels. Recent studies have demonstrated that KCHN1 mutations are implicated in Temple-Baraitser and Zimmermann-Laband syndromes and other forms of developmental deficits that all present with mental retardation and epilepsy, suggesting that Kv10.1 might be important for cognitive development in humans. Although the Kv10.1 channel is mainly expressed in the mammalian brain, its ectopic expression occurs in 70% of human cancers. Cancer cells and tumors expressing Kv10.1 acquire selective advantages that favor cancer progression through molecular mechanisms that involve several cellular pathways, indicating that protein-protein interactions may be important for Kv10.1 influence in cell proliferation and tumorigenesis. Several studies on transcriptional and post-transcriptional regulation of Kv10.1 expression have shown interesting mechanistic insights about Kv10.1 role in oncogenesis, increasing the importance of identifying the cellular factors that regulate Kv10.1 expression in tumors.

Lon in maintaining mitochondrial and endoplasmic reticulum homeostasis.

PubMed

Yang, Jieyeqi; Chen, Wenying; Zhang, Boyang; Tian, Fengli; Zhou, Zheng; Liao, Xin; Li, Chen; Zhang, Yi; Han, Yanyan; Wang, Yan; Li, Yuzhe; Wang, Guo-Qing; Shen, Xiao Li

2018-06-01

As a vital member of AAA+ (ATPase associated with diverse cellular activities) protein superfamily, Lon, a homo-hexameric ring-shaped protein complex with a serine-lysine catalytic dyad, is highly conserved throughout almost all prokaryotic and eukaryotic organisms. Lon protease (LONP) plays an important role in maintaining mitoproteostasis through selectively recognizing and degrading oxidatively modified mitoproteins within mitochondrial matrix, such as oxidized aconitase, phosphorylated mitochondrial transcription factor A, etc. Furthermore, the up-regulated LONP increased mitochondrial ROS generation to promote cell survival, cell proliferation, epithelial-mesenchymal transition, and cell migration, which was attributed to the up-regulation of NADH:ubiquinone oxidoreductase core subunit S8 via interaction with chaperone Lon under hypoxic or oxidative stress in tumorigenesis. In addition, Lon also participated in protein kinase RNA (PKR)-like endoplasmic reticulum kinase signaling pathway under endoplasmic reticulum (ER) stress. In short, Lon, as a pivotal stress-responsive protein that involved in the crosstalks among mitochondria, ER and nucleus, participated in multifarious important cellular processes crucial for cell survival, such as the mitochondrial protein quality control system, the mitochondrial unfolded protein response, the mtDNA maintenance, and the ER unfolded protein response.

Taming the Sphinx: Mechanisms of Cellular Sphingolipid Homeostasis

PubMed Central

Olson, D. K.; Fröhlich, F.; Farese, R; Walther, T. C.

2016-01-01

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. PMID:26747648

Fusion pores and their control of neurotransmitter and hormone release

PubMed Central

Chang, Che-Wei; Chiang, Chung-Wei

2017-01-01

Ca2+-triggered exocytosis functions broadly in the secretion of chemical signals, enabling neurons to release neurotransmitters and endocrine cells to release hormones. The biological demands on this process can vary enormously. Although synapses often release neurotransmitter in a small fraction of a millisecond, hormone release can be orders of magnitude slower. Vesicles usually contain multiple signaling molecules that can be released selectively and conditionally. Cells are able to control the speed, concentration profile, and content selectivity of release by tuning and tailoring exocytosis to meet different biological demands. Much of this regulation depends on the fusion pore—the aqueous pathway by which molecules leave a vesicle and move out into the surrounding extracellular space. Studies of fusion pores have illuminated how cells regulate secretion. Furthermore, the formation and growth of fusion pores serve as a readout for the progress of exocytosis, thus revealing key kinetic stages that provide clues about the underlying mechanisms. Herein, we review the structure, composition, and dynamics of fusion pores and discuss the implications for molecular mechanisms as well as for the cellular regulation of neurotransmitter and hormone release. PMID:28167663

Central thalamic deep brain stimulation for support of forebrain arousal regulation in the minimally conscious state.

PubMed

Schiff, Nicholas D

2013-01-01

This chapter considers the use of central thalamic deep brain stimulation (CT/DBS) to support arousal regulation mechanisms in the minimally conscious state (MCS). CT/DBS for selected patients in a MCS is first placed in the historical context of prior efforts to use thalamic electrical brain stimulation to treat the unconscious clinical conditions of coma and vegetative state. These previous studies and a proof of concept result from a single-subject study of a patient in a MCS are reviewed against the background of new population data providing benchmarks of the natural history of vegetative and MCSs. The conceptual foundations for CT/DBS in selected patients in a MCS are then presented with consideration of both circuit and cellular mechanisms underlying recovery of consciousness identified from empirical studies. Directions for developing future generalizable criteria for CT/DBS that focus on the integrity of necessary brain systems and behavioral profiles in patients in a MCS that may optimally response to support of arousal regulation mechanisms are proposed. © 2013 Elsevier B.V. All rights reserved.

Taming the sphinx: Mechanisms of cellular sphingolipid homeostasis.

PubMed

Olson, D K; Fröhlich, F; Farese, R V; Walther, T C

2016-08-01

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. Copyright © 2015. Published by Elsevier B.V.

Structure and Dynamic Regulation of Abl Kinases*

PubMed Central

Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Engen, John R.; Smithgall, Thomas E.

2013-01-01

The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity. PMID:23316053

Therapeutic strategies impacting cancer cell glutamine metabolism

PubMed Central

Lukey, Michael J; Wilson, Kristin F; Cerione, Richard A

2014-01-01

The metabolic adaptations that support oncogenic growth can also render cancer cells dependent on certain nutrients. Along with the Warburg effect, increased utilization of glutamine is one of the metabolic hallmarks of the transformed state. Glutamine catabolism is positively regulated by multiple oncogenic signals, including those transmitted by the Rho family of GTPases and by c-Myc. The recent identification of mechanistically distinct inhibitors of glutaminase, which can selectively block cellular transformation, has revived interest in the possibility of targeting glutamine metabolism in cancer therapy. Here, we outline the regulation and roles of glutamine metabolism within cancer cells and discuss possible strategies for, and the consequences of, impacting these processes therapeutically. PMID:24047273

Effect of posttranslational modifications on enzyme function and assembly.

PubMed

Ryšlavá, Helena; Doubnerová, Veronika; Kavan, Daniel; Vaněk, Ondřej

2013-10-30

The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

The role of CYP26 enzymes in retinoic acid clearance.

PubMed

Thatcher, Jayne E; Isoherranen, Nina

2009-08-01

Retinoic acid (RA) is a critical signaling molecule that regulates gene transcription and the cell cycle. Understanding of RA signaling has increased dramatically over the past decades, but the connection between whole body RA homeostasis and gene regulation in individual cells is still unclear. It has been proposed that cytochrome P450 family 26 (CYP26) enzymes have a role in determining the cellular exposure to RA by inactivating RA in cells that do not need RA. The CYP26 enzymes have been shown to metabolize RA efficiently and they are also inducible by RA in selected systems. However, their expression patterns in different cell types and a mechanistic understanding of their function is still lacking. Based on preliminary kinetic data and protein expression levels, one may predict that if CYP26A1 is expressed in the liver at even very low levels, it will be the major RA hydroxylase in this tissue. As such, it is an attractive pharmacological target for drug development when one aims to increase circulating or cellular RA concentrations. To further the understanding of how CYP26 enzymes contribute to the regulation of RA homeostasis, structural information of the CYP26s, commercially available recombinant enzymes and good specific and sensitive antibodies are needed.

The role of CYP26 enzymes in retinoic acid clearance

PubMed Central

Thatcher, Jayne E.; Isoherranen, Nina

2009-01-01

Retinoic acid (RA) is a critical signaling molecule that regulates gene transcription and the cell cycle. Understanding of RA signaling has increased dramatically over the past decades, but the connection between whole body RA homeostasis and gene regulation in individual cells is still unclear. It has been proposed that cytochrome P450 family 26 (CYP26) enzymes have a role in determining the cellular exposure to RA by inactivating RA in cells that do not need RA. The CYP26 enzymes have been shown to metabolize RA efficiently and they are also inducible by RA in selected systems. However, their expression patterns in different cell types and a mechanistic understanding of their function is still lacking. Based on preliminary kinetic data and protein expression levels, one may predict that if CYP26A1 is expressed in the liver at even very low levels, it will be the major RA hydroxylase in this tissue. As such, it is an attractive pharmacological target for drug development when one aims to increase circulating or cellular RA concentrations. To further the understanding of how CYP26 enzymes contribute to the regulation of RA homeostasis, structural information of the CYP26’s, commercially available recombinant enzymes and good specific and sensitive antibodies are needed. PMID:19519282

Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

PubMed Central

Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

2012-01-01

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

PubMed Central

Bruntz, Ronald C.; Lindsley, Craig W.

2014-01-01

Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein–coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. PMID:25244928

Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer.

PubMed

Bruntz, Ronald C; Lindsley, Craig W; Brown, H Alex

2014-10-01

Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein-coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Miklós Bodanszky Award Lecture: Advances in the selective targeting of protein phosphatase‐1 and phosphatase‐2A with peptides

PubMed Central

2017-01-01

Protein phosphatase‐1 and phosphatase‐2A are two ubiquitously expressed enzymes known to catalyze the majority of dephosphorylation reactions on serine and threonine inside cells. They play roles in most cellular processes and are tightly regulated by regulatory subunits in holoenzymes. Their misregulation and malfunction contribute to disease development and progression, such as in cancer, diabetes, viral infections, and neurological as well as heart diseases. Therefore, targeting these phosphatases for therapeutic use would be highly desirable; however, their complex regulation and high conservation of the active site have been major hurdles for selectively targeting them in the past. In the last decade, new approaches have been developed to overcome these hurdles and have strongly revived the field. I will focus here on peptide‐based approaches, which contributed to showing that these phosphatases can be targeted selectively and aided in rethinking the design of selective phosphatase modulators. Finally, I will give a perspective on www.depod.org, the human dephosphorylation database, and how it can aid phosphatase modulator design. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. PMID:28876538

Cellular copper homeostasis: current concepts on its interplay with glutathione homeostasis and its implication in physiology and human diseases.

PubMed

Bhattacharjee, Ashima; Chakraborty, Kaustav; Shukla, Aditya

2017-10-18

Copper is a trace element essential for almost all living organisms. But the level of intracellular copper needs to be tightly regulated. Dysregulation of cellular copper homeostasis leading to various diseases demonstrates the importance of this tight regulation. Copper homeostasis is regulated not only within the cell but also within individual intracellular compartments. Inactivation of export machinery results in excess copper being redistributed into various intracellular organelles. Recent evidence suggests the involvement of glutathione in playing an important role in regulating copper entry and intracellular copper homeostasis. Therefore interplay of both homeostases might play an important role within the cell. Similar to copper, glutathione balance is tightly regulated within individual cellular compartments. This review explores the existing literature on the role of glutathione in regulating cellular copper homeostasis. On the one hand, interplay of glutathione and copper homeostasis performs an important role in normal physiological processes, for example neuronal differentiation. On the other hand, perturbation of the interplay might play a key role in the pathogenesis of copper homeostasis disorders.

Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide

PubMed Central

Fomenko, Dmitri E.; Koc, Ahmet; Agisheva, Natalia; Jacobsen, Michael; Kaya, Alaattin; Malinouski, Mikalai; Rutherford, Julian C.; Siu, Kam-Leung; Jin, Dong-Yan; Winge, Dennis R.; Gladyshev, Vadim N.

2011-01-01

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H2O2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H2O2. Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H2O2. The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H2O2 and other cellular proteins plays a secondary role. PMID:21282621

Regulation of cellular senescence by the essential caveolar component PTRF/Cavin-1

PubMed Central

Bai, Lin; Deng, Xiaoli; Li, Juanjuan; Wang, Miao; Li, Qian; An, Wei; A, Deli; Cong, Yu-Sheng

2011-01-01

Polymerase I and transcript release factor (PTRF, also known as Cavin-1) is an essential component in the biogenesis and function of caveolae. Here, we show that PTRF expression is increased in senescent human fibroblasts. Importantly, overexpression of PTRF induced features characteristic of cellular senescence, whereas reduced PTRF expression extended the cellular replicative lifespan. Interestingly, we found that PTRF localized primarily to the nuclei of young and quiescent WI-38 human fibroblasts, but translocated to the cytosol and plasma membrane during cellular senescence. Furthermore, electron microscopic analysis demonstrated an increased number of caveolar structures in senescent and PTRF-transfected WI-38 cells. Our data suggest that the role of PTRF in cellular senescence is dependent on its targeting to caveolae and its interaction with caveolin-1, which appeared to be regulated by the phosphorylation of PTRF. Taken together, our findings identify PTRF as a novel regulator of cellular senescence that acts through the p53/p21 and caveolar pathways. PMID:21445100

Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium

PubMed Central

Delos Santos, Ralph Christian; Bautista, Stephen; Lucarelli, Stefanie; Bone, Leslie N.; Dayam, Roya M.; Abousawan, John; Botelho, Roberto J.; Antonescu, Costin N.

2017-01-01

Clathrin-mediated endocytosis is a major regulator of cell-surface protein internalization. Clathrin and other proteins assemble into small invaginating structures at the plasma membrane termed clathrin-coated pits (CCPs) that mediate vesicle formation. In addition, epidermal growth factor receptor (EGFR) signaling is regulated by its accumulation within CCPs. Given the diversity of proteins regulated by clathrin-mediated endocytosis, how this process may distinctly regulate specific receptors is a key question. We examined the selective regulation of clathrin-dependent EGFR signaling and endocytosis. We find that perturbations of phospholipase Cγ1 (PLCγ1), Ca2+, or protein kinase C (PKC) impair clathrin-mediated endocytosis of EGFR, the formation of CCPs harboring EGFR, and EGFR signaling. Each of these manipulations was without effect on the clathrin-mediated endocytosis of transferrin receptor (TfR). EGFR and TfR were recruited to largely distinct clathrin structures. In addition to control of initiation and assembly of CCPs, EGF stimulation also elicited a Ca2+- and PKC-dependent reduction in synaptojanin1 recruitment to clathrin structures, indicating broad control of CCP assembly by Ca2+ signals. Hence EGFR elicits PLCγ1-calcium signals to facilitate formation of a subset of CCPs, thus modulating its own signaling and endocytosis. This provides evidence for the versatility of CCPs to control diverse cellular processes. PMID:28814502

Phosphatidylinositol transfer protein beta displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin.

PubMed

Ségui, Bruno; Allen-Baume, Victoria; Cockcroft, Shamshad

2002-08-15

Mammalian phosphatidylinositol transfer proteins (PITPs) alpha and beta, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities. In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPbeta has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPbeta is important for the regulation of SM metabolism. In the present study, we have analysed the ability of human PITPbeta to transfer and regulate the metabolism of cellular SM. We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer. Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPbeta being slightly greater. To identify which cellular lipids were selected by PITPs, PITPalpha and PITPbeta were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids. Both PITPs were able to select only PI and PC, but not SM. SM synthesis takes place at the Golgi, and PITPbeta was shown to localize in that compartment. To examine the role of PITPbeta in SM biosynthesis, Golgi membranes were used. Purified Golgi membranes had lost their endogenous PITPbeta, but were able to recruit PITPbeta when added exogenously. However, PITPbeta did not enhance the activities of either SM synthase or glucosylceramide synthase. Further analysis in COS-7 cells overexpressing PITPbeta showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment. Altogether, these observations do not support the suggestion that PITPbeta participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking.

A pilgrim's progress: Seeking meaning in primordial germ cell migration.

PubMed

Cantú, Andrea V; Laird, Diana J

2017-10-01

Comparative studies of primordial germ cell (PGC) development across organisms in many phyla reveal surprising diversity in the route of migration, timing and underlying molecular mechanisms, suggesting that the process of migration itself is conserved. However, beyond the perfunctory transport of cellular precursors to their later arising home of the gonads, does PGC migration serve a function? Here we propose that the process of migration plays an additional role in quality control, by eliminating PGCs incapable of completing migration as well as through mechanisms that favor PGCs capable of responding appropriately to migration cues. Focusing on PGCs in mice, we explore evidence for a selective capacity of migration, considering the tandem regulation of proliferation and migration, cell-intrinsic and extrinsic control, the potential for tumors derived from failed PGC migrants, the potential mechanisms by which migratory PGCs vary in their cellular behaviors, and corresponding effects on development. We discuss the implications of a selective role of PGC migration for in vitro gametogenesis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Epithelial adhesion molecules and the regulation of intestinal homeostasis during neutrophil transepithelial migration

PubMed Central

Sumagin, Ronen; Parkos, Charles A

2014-01-01

Epithelial adhesion molecules play essential roles in regulating cellular function and maintaining mucosal tissue homeostasis. Some form epithelial junctional complexes to provide structural support for epithelial monolayers and act as a selectively permeable barrier separating luminal contents from the surrounding tissue. Others serve as docking structures for invading viruses and bacteria, while also regulating the immune response. They can either obstruct or serve as footholds for the immune cells recruited to mucosal surfaces. Currently, it is well appreciated that adhesion molecules collectively serve as environmental cue sensors and trigger signaling events to regulate epithelial function through their association with the cell cytoskeleton and various intracellular adapter proteins. Immune cells, particularly neutrophils (PMN) during transepithelial migration (TEM), can modulate adhesion molecule expression, conformation, and distribution, significantly impacting epithelial function and tissue homeostasis. This review discusses the roles of key intestinal epithelial adhesion molecules in regulating PMN trafficking and outlines the potential consequences on epithelial function. PMID:25838976

Recent Progress on Liver Kinase B1 (LKB1): Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

PubMed Central

Gan, Ren-You; Li, Hua-Bin

2014-01-01

Liver kinase B1 (LKB1), known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK)-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK), salt-inducible kinase (SIK), sucrose non-fermenting protein-related kinase (SNRK) and brain selective kinase (BRSK) signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers. PMID:25244018

Lipids, lysosomes, and autophagy

PubMed Central

2016-01-01

Lipids are essential components of a cell providing energy substrates for cellular processes, signaling intermediates, and building blocks for biological membranes. Lipids are constantly recycled and redistributed within a cell. Lysosomes play an important role in this recycling process that involves the recruitment of lipids to lysosomes via autophagy or endocytosis for their degradation by lysosomal hydrolases. The catabolites produced are redistributed to various cellular compartments to support basic cellular function. Several studies demonstrated a bidirectional relationship between lipids and lysosomes that regulate autophagy. While lysosomal degradation pathways regulate cellular lipid metabolism, lipids also regulate lysosome function and autophagy. In this review, we focus on this bidirectional relationship in the context of dietary lipids and provide an overview of recent evidence of how lipid-overload lipotoxicity, as observed in obesity and metabolic syndrome, impairs lysosomal function and autophagy that may eventually lead to cellular dysfunction or cell death. PMID:27330054

The Transcription Factor EB Links Cellular Stress to the Immune Response



PubMed Central

Nabar, Neel R.; Kehrl, John H.

2017-01-01

The transcription factor EB (TFEB) is the master transcriptional regulator of autophagy and lysosome biogenesis. Recent advances have led to a paradigm shift in our understanding of lysosomes from a housekeeping cellular waste bin to a dynamically regulated pathway that is efficiently turned up or down based on cellular needs. TFEB coordinates the cellular response to nutrient deprivation and other forms of cell stress through the lysosome system, and regulates a myriad of cellular processes associated with this system including endocytosis, phagocytosis, autophagy, and lysosomal exocytosis. Autophagy and the endolysosomal system are critical to both the innate and adaptive arms of the immune system, with functions in effector cell priming and direct pathogen clearance. Recent studies have linked TFEB to the regulation of the immune response through the endolysosmal pathway and by direct transcriptional activation of immune related genes. In this review, we discuss the current understanding of TFEB’s function and the molecular mechanisms behind TFEB activation. Finally, we discuss recent advances linking TFEB to the immune response that positions lysosomal signaling as a potential target for immune modulation. PMID:28656016

The Transcription Factor EB Links Cellular Stress to the Immune Response

.

PubMed

Nabar, Neel R; Kehrl, John H

2017-06-01

The transcription factor EB (TFEB) is the master transcriptional regulator of autophagy and lysosome biogenesis. Recent advances have led to a paradigm shift in our understanding of lysosomes from a housekeeping cellular waste bin to a dynamically regulated pathway that is efficiently turned up or down based on cellular needs. TFEB coordinates the cellular response to nutrient deprivation and other forms of cell stress through the lysosome system, and regulates a myriad of cellular processes associated with this system including endocytosis, phagocytosis, autophagy, and lysosomal exocytosis. Autophagy and the endolysosomal system are critical to both the innate and adaptive arms of the immune system, with functions in effector cell priming and direct pathogen clearance. Recent studies have linked TFEB to the regulation of the immune response through the endolysosmal pathway and by direct transcriptional activation of immune related genes. In this review, we discuss the current understanding of TFEB's function and the molecular mechanisms behind TFEB activation. Finally, we discuss recent advances linking TFEB to the immune response that positions lysosomal signaling as a potential target for immune modulation.

Cellular pressure and volume regulation and implications for cell mechanics

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean

2013-03-01

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death and migration. Volume and shape regulation also directly impacts the mechanics of the cell and multi-cellular tissues. Recent experiments found that during mitosis, eukaryotic cells establish a preferred steady volume and pressure, and the steady volume and pressure can robustly adapt to large osmotic shocks. Here we develop a mathematical model of cellular pressure and volume regulation, incorporating essential elements such as water permeation, mechano-sensitive channels, active ion pumps and active stresses in the actomyosin cortex. The model can fully explain the available experimental data, and predicts the cellular volume and pressure for several models of cell cortical mechanics. Furthermore, we show that when cells are subjected to an externally applied load, such as in an AFM indentation experiment, active regulation of volume and pressure leads to complex cellular response. We found the cell stiffness highly depends on the loading rate, which indicates the transport of water and ions might contribute to the observed viscoelasticity of cells.

Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue

PubMed Central

Smith, Carolyn L.; Abdallah, Salsabil; Le, Phuong; Harracksingh, Alicia N.; Artinian, Liana; Tamvacakis, Arianna N.; Rehder, Vincent; Reese, Thomas S.

2017-01-01

Four-domain voltage-gated Ca2+ (Cav) channels play fundamental roles in the nervous system, but little is known about when or how their unique properties and cellular roles evolved. Of the three types of metazoan Cav channels, Cav1 (L-type), Cav2 (P/Q-, N- and R-type) and Cav3 (T-type), Cav3 channels are optimized for regulating cellular excitability because of their fast kinetics and low activation voltages. These same properties permit Cav3 channels to drive low-threshold exocytosis in select neurons and neurosecretory cells. Here, we characterize the single T-type calcium channel from Trichoplax adhaerens (TCav3), an early diverging animal that lacks muscle, neurons, and synapses. Co-immunolocalization using antibodies against TCav3 and neurosecretory cell marker complexin labeled gland cells, which are hypothesized to play roles in paracrine signaling. Cloning and in vitro expression of TCav3 reveals that, despite roughly 600 million years of divergence from other T-type channels, it bears the defining structural and biophysical features of the Cav3 family. We also characterize the channel’s cation permeation properties and find that its pore is less selective for Ca2+ over Na+ compared with the human homologue Cav3.1, yet it exhibits a similar potent block of inward Na+ current by low external Ca2+ concentrations (i.e., the Ca2+ block effect). A comparison of the permeability features of TCav3 with other cloned channels suggests that Ca2+ block is a locus of evolutionary change in T-type channel cation permeation properties and that mammalian channels distinguish themselves from invertebrate ones by bearing both stronger Ca2+ block and higher Ca2+ selectivity. TCav3 is the most divergent metazoan T-type calcium channel and thus provides an evolutionary perspective on Cav3 channel structure–function properties, ion selectivity, and cellular physiology. PMID:28330839

The Predator becomes the Prey: Regulating the Ubiquitin System by Ubiquitylation and Degradation

PubMed Central

Weissman, Allan M.; Shabek, Nitzan; Ciechanover, Aaron

2012-01-01

Ubiquitylation (also known as ubiquitination) regulates essentially all intracellular processes in eukaryotes through highly specific, and often tightly spatially and temporally regulated, modification of numerous cellular proteins. Although most often associated with proteasomal degradation, ubiquitylation frequently serves non-proteolytic functions. In light of its central roles in cellular regulation, it has not been surprising to find that many of the components of the ubiquitin system itself are regulated by ubiquitylation. This observation has broad implications for pathophysiology. PMID:21860393

Regulation of nitrate assimilation in cyanobacteria.

PubMed

Ohashi, Yoshitake; Shi, Wei; Takatani, Nobuyuki; Aichi, Makiko; Maeda, Shin-ichi; Watanabe, Satoru; Yoshikawa, Hirofumi; Omata, Tatsuo

2011-02-01

Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

PubMed Central

Hristov, Kiril L.; Smith, Amy C.; Parajuli, Shankar P.; Malysz, John

2013-01-01

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility. PMID:24352333

Cardiac Epithelial-Mesenchymal Transition Is Blocked by Monomethylarsonous Acid (III)

PubMed Central

Huang, Tianfang; Barnett, Joey V.; Camenisch, Todd D.

2014-01-01

Arsenic exposure during embryonic development can cause ischemic heart pathologies later in adulthood which may originate from impairment in proper blood vessel formation. The arsenic-associated detrimental effects are mediated by arsenite (iAsIII) and its most toxic metabolite, monomethylarsonous acid [MMA (III)]. The impact of MMA (III) on coronary artery development has not yet been studied. The key cellular process that regulates coronary vessel development is the epithelial-mesenchymal transition (EMT). During cardiac EMT, activated epicardial progenitor cells transform to mesenchymal cells to form the cellular components of coronary vessels. Smad2/3 mediated TGFβ2 signaling, the key regulator of cardiac EMT, is disrupted by arsenite exposure. In this study, we compared the cardiac toxicity of MMA (III) with arsenite. Epicardial progenitor cells are 15 times more sensitive to MMA (III) cytotoxicity when compared with arsenite. MMA (III) caused a significant blockage in epicardial cellular transformation and invasion at doses 10 times lower than arsenite. Key EMT genes including TGFβ ligands, TβRIII, Has2, CD44, Snail1, TBX18, and MMP2 were down regulated by MMA (III) exposure. MMA (III) disrupted Smad2/3 activation at a dose 20 times lower than arsenite. Both arsenite and MMA (III) significantly inhibited Erk1/2 and Erk5 phosphorylation. Nuclear translocation of Smad2/3 and Erk5 was also blocked by arsenical exposure. However, p38 activation, as well as smooth muscle differentiation, was refractory to the inhibition by the arsenicals. Collectively, these findings revealed that MMA (III) is a selective disruptor of cardiac EMT and as such may predispose to arsenic-associated cardiovascular disorders. PMID:25145660

Gold Nanoparticles for Biology and Medicine

PubMed Central

Giljohann, David A.; Seferos, Dwight S.; Daniel, Weston L.; Massich, Matthew D.; Patel, Pinal C.

2014-01-01

Gold colloids have fascinated scientists for over a century and are now heavily utilized in chemistry, biology, engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to molecule-based systems, but rather for their new physical and chemical properties, which confer substantive advantages in cellular and medical applications. PMID:20401880

U1snRNP-mediated suppression of polyadenylation in conjunction with the RNA structure controls poly (A) site selection in foamy viruses

PubMed Central

2013-01-01

Background During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5′LTR and activate polyadenylation in the 3′LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal. Results Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5′LTR. In contrast, polyadenylation at the 3′LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation. Conclusion Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5’end of their RNA. At the 3’end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression. PMID:23718736

Charge Inversion in semi-permeable membranes

NASA Astrophysics Data System (ADS)

Das, Siddhartha; Sinha, Shayandev; Jing, Haoyuan

Role of semi-permeable membranes like lipid bilayer is ubiquitous in a myriad of physiological and pathological phenomena. Typically, lipid membranes are impermeable to ions and solutes; however, protein channels embedded in the membrane allow the passage of selective, small ions across the membrane enabling the membrane to adopt a semi-permeable nature. This semi-permeability, in turn, leads to electrostatic potential jump across the membrane, leading to effects such as regulation of intracellular calcium, extracellular-vesicle-membrane interactions, etc. In this study, we theoretically demonstrate that this semi-permeable nature may trigger the most remarkable charge inversion (CI) phenomenon in the cytosol-side of the negatively-charged lipid bilayer membrane that are selectively permeable to only positive ions of a given salt. This CI is manifested as the changing of the sign of the electrostatic potential from negative to positive from the membrane-cytosol interface to deep within the cytosol. We study the impact of the parameters such as the concentration of this salt with selectively permeable ions as well as the concentration of an external salt in the development of this CI phenomenon. We anticipate such CI will profoundly influence the interaction of membrane and intra-cellular moieties (e.g., exosome or multi-cellular vesicles) having implications for a host of biophysical processes.

Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

PubMed Central

Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

2014-01-01

Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. PMID:25305018

A chitinase from pacific white shrimp Litopenaeus vannamei involved in immune regulation.

PubMed

Niu, Shengwen; Yang, Linwei; Zuo, Hongliang; Zheng, Jiefu; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

2018-08-01

Chitinases are a group of hydrolytic enzymes that hydrolyze chitin and widely exist in organisms. Studies in mammals have demonstrated that chitinases play important roles in regulation of humoral and cellular immune responses. In arthropods, although it is well known that chitinases are involved in growth, molting and development, the current knowledge on the role of chitinases in immunity, especially in immune regulation, remains largely unknown. In this study, a chitinase (LvChi5) from Litopenaeus vannamei was representatively selected for studying its immune function. The start codon of LvChi5 was corrected by 5'RACE analysis and its protein sequence was reanalyzed. LvChi5 contains a catalytic domain and a chitin binding domain and shows no inhibitory effect on growth of bacteria in vitro. However, in vivo experiments demonstrated that silencing of LvChi5 increased the mortality of shrimp infected with white spot syndrome virus (WSSV) and Vibro parahaemolyticus and significantly upregulated the load of pathogens in tissues. The expression of various immune related genes, including transcription factors, antimicrobial peptides and other functional proteins with antibacterial and antiviral activities, was widely changed in LvChi5 silencing shrimp. Moreover, the recombinant LvChi5 protein could enhance the phagocytic activity of hemocytes against bacteria. These suggested that shrimp chitinase could play a role in regulation of both humoral and cellular immune responses in shrimp. Copyright © 2018 Elsevier Ltd. All rights reserved.

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters.

PubMed

Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

2012-02-01

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks.

PubMed

Slawson, Chad; Housley, Michael P; Hart, Gerald W

2006-01-01

O-GlcNAc is an ubiquitous post-translational protein modification consisting of a single N-acetlyglucosamine moiety linked to serine or threonine residues on nuclear and cytoplasmic proteins. Recent work has begun to uncover the functional roles of O-GlcNAc in cellular processes. O-GlcNAc modified proteins are involved in sensing the nutrient status of the surrounding cellular environment and adjusting the activity of cellular proteins accordingly. O-GlcNAc regulates cellular responses to hormones such as insulin, initiates a protective response to stress, modulates a cell's capacity to grow and divide, and regulates gene transcription. This review will focus on recent work involving O-GlcNAc in sensing the environment and regulating signaling cascades. (c) 2005 Wiley-Liss, Inc.

Mitochondria targeting by environmental stressors: Implications for redox cellular signaling.

PubMed

Blajszczak, Chuck; Bonini, Marcelo G

2017-11-01

Mitochondria are cellular powerhouses as well as metabolic and signaling hubs regulating diverse cellular functions, from basic physiology to phenotypic fate determination. It is widely accepted that reactive oxygen species (ROS) generated in mitochondria participate in the regulation of cellular signaling, and that some mitochondria chronically operate at a high ROS baseline. However, it is not completely understood how mitochondria adapt to persistently high ROS states and to environmental stressors that disturb the redox balance. Here we will review some of the current concepts regarding how mitochondria resist oxidative damage, how they are replaced when excessive oxidative damage compromises function, and the effect of environmental toxicants (i.e. heavy metals) on the regulation of mitochondrial ROS (mtROS) production and subsequent impact. Copyright © 2017 Elsevier B.V. All rights reserved.

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

PubMed

Sarkar, Anwesha; Zhao, Yuanchang; Wang, Yongliang; Wang, Xuefeng

2018-06-25

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging. The FAC molecule consists of an adhesive domain for surface coating and a force-reporting domain which can be activated to fluoresce by integrin molecular tension. The tension threshold required for FAC activation is tunable in 10-60 piconewton (pN), allowing the selective imaging of cellular force contributed by integrin tension at different force levels. We tested the performance of two FACs with tension thresholds of 12 and 54 pN (nominal values), respectively, on both glass and polystyrene surfaces. Cellular forces were successfully mapped by fluorescence imaging on all the surfaces. FAC-coated surfaces also enable co-imaging of cellular forces and cell structures in both live cells and immunostained cells, therefore opening a new avenue for the study of the interplay of force and structure. We demonstrated the co-imaging of integrin tension and talin clustering in live cells, and concluded that talin clustering always occurs before the generation of integrin tension above 54 pN, reinforcing the notion that talin is an important adaptor protein for integrin tension transmission. Overall, FAC provides a highly convenient approach that is accessible to general biological laboratories for the study of cellular forces with high sensitivity and resolution, thus holding the potential to greatly boost the research of cell mechanobiology.

Physiology and Evolution of Voltage-Gated Calcium Channels in Early Diverging Animal Phyla: Cnidaria, Placozoa, Porifera and Ctenophora

PubMed Central

Senatore, Adriano; Raiss, Hamad; Le, Phuong

2016-01-01

Voltage-gated calcium (Cav) channels serve dual roles in the cell, where they can both depolarize the membrane potential for electrical excitability, and activate transient cytoplasmic Ca2+ signals. In animals, Cav channels play crucial roles including driving muscle contraction (excitation-contraction coupling), gene expression (excitation-transcription coupling), pre-synaptic and neuroendocrine exocytosis (excitation-secretion coupling), regulation of flagellar/ciliary beating, and regulation of cellular excitability, either directly or through modulation of other Ca2+-sensitive ion channels. In recent years, genome sequencing has provided significant insights into the molecular evolution of Cav channels. Furthermore, expanded gene datasets have permitted improved inference of the species phylogeny at the base of Metazoa, providing clearer insights into the evolution of complex animal traits which involve Cav channels, including the nervous system. For the various types of metazoan Cav channels, key properties that determine their cellular contribution include: Ion selectivity, pore gating, and, importantly, cytoplasmic protein-protein interactions that direct sub-cellular localization and functional complexing. It is unclear when these defining features, many of which are essential for nervous system function, evolved. In this review, we highlight some experimental observations that implicate Cav channels in the physiology and behavior of the most early-diverging animals from the phyla Cnidaria, Placozoa, Porifera, and Ctenophora. Given our limited understanding of the molecular biology of Cav channels in these basal animal lineages, we infer insights from better-studied vertebrate and invertebrate animals. We also highlight some apparently conserved cellular functions of Cav channels, which might have emerged very early on during metazoan evolution, or perhaps predated it. PMID:27867359

The molecular mechanisms of plant plasma membrane intrinsic proteins trafficking and stress response.

PubMed

Wang, Xing; Zhang, Ji-long; Feng, Xiu-xiu; Li, Hong-jie; Zhang, Gen-fa

2017-04-20

Plasma membrane intrinsic proteins (PIPs) are plant channel proteins located on the plasma membrane. PIPs transfer water, CO 2 and small uncharged solutes through the plasma membrane. PIPs have high selectivity to substrates, suggestive of a central role in maintaining cellular water balance. The expression, activity and localization of PIPs are regulated at the transcriptional and post-translational levels, and also affected by environmental factors. Numerous studies indicate that the expression patterns and localizations of PIPs can change in response to abiotic stresses. In this review, we summarize the mechanisms of PIP trafficking, transcriptional and post-translational regulations, and abiotic stress responses. Moreover, we also discuss the current research trends and future directions on PIPs.

AMP-Activated Protein Kinase: An Ubiquitous Signaling Pathway With Key Roles in the Cardiovascular System.

PubMed

Salt, Ian P; Hardie, D Grahame

2017-05-26

The AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis, which acts to restore energy homoeostasis whenever cellular energy charge is depleted. Over the last 2 decades, it has become apparent that AMPK regulates several other cellular functions and has specific roles in cardiovascular tissues, acting to regulate cardiac metabolism and contractile function, as well as promoting anticontractile, anti-inflammatory, and antiatherogenic actions in blood vessels. In this review, we discuss the role of AMPK in the cardiovascular system, including the molecular basis of mutations in AMPK that alter cardiac physiology and the proposed mechanisms by which AMPK regulates vascular function under physiological and pathophysiological conditions. © 2017 American Heart Association, Inc.

Selective Cooperation between Fatty Acid Binding Proteins and Peroxisome Proliferator-Activated Receptors in Regulating Transcription

PubMed Central

Tan, Nguan-Soon; Shaw, Natacha S.; Vinckenbosch, Nicolas; Liu, Peng; Yasmin, Rubina; Desvergne, Béatrice; Wahli, Walter; Noy, Noa

2002-01-01

Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARγ and PPARβ, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARγ and PPARβ and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARβ-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions. PMID:12077340

Distinct Mechanisms of Pathogenic DJ-1 Mutations in Mitochondrial Quality Control

PubMed Central

Strobbe, Daniela; Robinson, Alexis A.; Harvey, Kirsten; Rossi, Lara; Ferraina, Caterina; de Biase, Valerio; Rodolfo, Carlo; Harvey, Robert J.; Campanella, Michelangelo

2018-01-01

The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson’s disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein–protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1D149A mutant does not influence mitophagy, but instead impairs Ca2+ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis. PMID:29599708

Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles

PubMed Central

Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

2016-01-01

Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers. PMID:27531648

Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles

NASA Astrophysics Data System (ADS)

Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

2016-08-01

Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers.

Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles.

PubMed

Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

2016-08-17

Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers.

Rab GTPases in Immunity and Inflammation.

PubMed

Prashar, Akriti; Schnettger, Laura; Bernard, Elliott M; Gutierrez, Maximiliano G

2017-01-01

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

The role of focal adhesion kinase in the regulation of cellular mechanical properties

NASA Astrophysics Data System (ADS)

Mierke, Claudia Tanja

2013-12-01

The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation.

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

PubMed

Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Geo, Jini; Dilipkumar, Shilpa; Pasricha, Renu; Gulyani, Akash; Raghavan, Srikala; Palakodeti, Dasaradhi

2017-09-01

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1 , are translationally regulated by SMED-PABPC2 . Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. © 2017. Published by The Company of Biologists Ltd.

Ceramide induced mitophagy and tumor suppression

PubMed Central

Dany, Mohammed; Ogretmen, Besim

2015-01-01

Sphingolipids are bioactive lipid effectors, which are involved in the regulation of various cellular signaling pathways. Sphingolipids play essential roles in controlling cell inflammation, proliferation, death, migration, senescence, metastasis and autophagy. Alterations in sphingolipid metabolism has been also implicated in many human cancers. Macroautophagy (referred to here as autophagy) is a form of nonselective sequestering of cytosolic materials by double membrane structures, autophagosomes, which can be either protective or lethal for cells. Ceramide, a central molecule of sphingolipid metabolism is involved in the regulation of autophagy at various levels, including the induction of lethal mitophagy, a selective autophagy process to target and eliminate damaged mitochondria. In this review, we focused on recent studies with regard to the regulation of autophagy, in particular lethal mitophagy, by ceramide, and aimed at providing discussion points for various context-dependent roles and mechanisms of action of ceramide in controlling mitophagy. PMID:25634657

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea

PubMed Central

Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Dilipkumar, Shilpa; Gulyani, Akash; Raghavan, Srikala

2017-01-01

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. PMID:28807897

Altered interactions between unicellular and multicellular genes drive hallmarks of transformation in a diverse range of solid tumors.

PubMed

Trigos, Anna S; Pearson, Richard B; Papenfuss, Anthony T; Goode, David L

2017-06-13

Tumors of distinct tissues of origin and genetic makeup display common hallmark cellular phenotypes, including sustained proliferation, suppression of cell death, and altered metabolism. These phenotypic commonalities have been proposed to stem from disruption of conserved regulatory mechanisms evolved during the transition to multicellularity to control fundamental cellular processes such as growth and replication. Dating the evolutionary emergence of human genes through phylostratigraphy uncovered close association between gene age and expression level in RNA sequencing data from The Cancer Genome Atlas for seven solid cancers. Genes conserved with unicellular organisms were strongly up-regulated, whereas genes of metazoan origin were primarily inactivated. These patterns were most consistent for processes known to be important in cancer, implicating both selection and active regulation during malignant transformation. The coordinated expression of strongly interacting multicellularity and unicellularity processes was lost in tumors. This separation of unicellular and multicellular functions appeared to be mediated by 12 highly connected genes, marking them as important general drivers of tumorigenesis. Our findings suggest common principles closely tied to the evolutionary history of genes underlie convergent changes at the cellular process level across a range of solid cancers. We propose altered activity of genes at the interfaces between multicellular and unicellular regions of human gene regulatory networks activate primitive transcriptional programs, driving common hallmark features of cancer. Manipulation of cross-talk between biological processes of different evolutionary origins may thus present powerful and broadly applicable treatment strategies for cancer.

Altered interactions between unicellular and multicellular genes drive hallmarks of transformation in a diverse range of solid tumors

PubMed Central

Trigos, Anna S.; Pearson, Richard B.; Papenfuss, Anthony T.; Goode, David L.

2017-01-01

Tumors of distinct tissues of origin and genetic makeup display common hallmark cellular phenotypes, including sustained proliferation, suppression of cell death, and altered metabolism. These phenotypic commonalities have been proposed to stem from disruption of conserved regulatory mechanisms evolved during the transition to multicellularity to control fundamental cellular processes such as growth and replication. Dating the evolutionary emergence of human genes through phylostratigraphy uncovered close association between gene age and expression level in RNA sequencing data from The Cancer Genome Atlas for seven solid cancers. Genes conserved with unicellular organisms were strongly up-regulated, whereas genes of metazoan origin were primarily inactivated. These patterns were most consistent for processes known to be important in cancer, implicating both selection and active regulation during malignant transformation. The coordinated expression of strongly interacting multicellularity and unicellularity processes was lost in tumors. This separation of unicellular and multicellular functions appeared to be mediated by 12 highly connected genes, marking them as important general drivers of tumorigenesis. Our findings suggest common principles closely tied to the evolutionary history of genes underlie convergent changes at the cellular process level across a range of solid cancers. We propose altered activity of genes at the interfaces between multicellular and unicellular regions of human gene regulatory networks activate primitive transcriptional programs, driving common hallmark features of cancer. Manipulation of cross-talk between biological processes of different evolutionary origins may thus present powerful and broadly applicable treatment strategies for cancer. PMID:28484005

Regulatory Myeloid Cells in Transplantation

PubMed Central

Rosborough, Brian R.; Raïch-Regué, Dàlia; Turnquist, Heth R.; Thomson, Angus W.

2013-01-01

Regulatory myeloid cells (RMC) are emerging as novel targets for immunosuppressive (IS) agents and hold considerable promise as cellular therapeutic agents. Herein, we discuss the ability of regulatory macrophages (Mreg), regulatory dendritic cells (DCreg) and myeloid-derived suppressor cells (MDSC) to regulate alloimmunity, their potential as cellular therapeutic agents and the IS agents that target their function. We consider protocols for the generation of RMC and the selection of donor- or recipient-derived cells for adoptive cell therapy. Additionally, the issues of cell trafficking and antigen (Ag) specificity following RMC transfer are discussed. Improved understanding of the immunobiology of these cells has increased the possibility of moving RMC into the clinic to reduce the burden of current IS agents and promote Ag-specific tolerance. In the second half of this review, we discuss the influence of established and experimental IS agents on myeloid cell populations. IS agents believed historically to act primarily on T cell activation and proliferation are emerging as important regulators of RMC function. Better insights into the influence of IS agents on RMC will enhance our ability to develop cell therapy protocols to promote the function of these cells. Moreover, novel IS agents may be designed to target RMC in situ to promote Ag-specific immune regulation in transplantation and usher in a new era of immune modulation exploiting cells of myeloid origin. PMID:24092382

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

PubMed Central

Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K.; Arora, Karunesh; Brooks, Charles L.; Raghavan, Malini

2015-01-01

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics. PMID:26420867

Linking the Primary Cilium to Cell Migration in Tissue Repair and Brain Development

PubMed Central

Veland, Iben Rønn; Lindbæk, Louise; Christensen, Søren Tvorup

2014-01-01

Primary cilia are unique sensory organelles that coordinate cellular signaling networks in vertebrates. Inevitably, defects in the formation or function of primary cilia lead to imbalanced regulation of cellular processes that causes multisystemic disorders and diseases, commonly known as ciliopathies. Mounting evidence has demonstrated that primary cilia coordinate multiple activities that are required for cell migration, which, when they are aberrantly regulated, lead to defects in organogenesis and tissue repair, as well as metastasis of tumors. Here, we present an overview on how primary cilia may contribute to the regulation of the cellular signaling pathways that control cyclic processes in directional cell migration. PMID:26955067

The Novel Poly(A) Polymerase Star-PAP is a Signal-Regulated Switch at the 3′-end of mRNAs

PubMed Central

Li, Weimin; Laishram, Rakesh S.; Anderson, Richard A.

2013-01-01

The mRNA 3′-untranslated region (3′-UTR) modulates message stability, transport, intracellular location and translation. We have discovered a novel nuclear poly(A) polymerase termed Star-PAP (nuclear speckle targeted PIPKIα regulated-poly(A) polymerase) that couples with the transcriptional machinery and is regulated by the phosphoinositide lipid messenger phosphatidylinositol-4,5-bisphosphate (PI4,5P2), the central lipid in phosphoinositide signaling. PI4,5P2 is generated primarily by type I phosphatidylinositol phosphate kinases (PIPKI). Phosphoinositides are present in the nucleus including at nuclear speckles compartments separate from known membrane structures. PIPKs regulate cellular functions by interacting with PI4,5P2 effectors where PIPKs generate PI4,5P2 that then modulates the activity of the associated effectors. Nuclear PIPKIα interacts with and regulates Star-PAP, and PI4,5P2 specifically activates Star-PAP in a gene- and signaling-dependent manner. Importantly, other select signaling molecules integrated into the Star-PAP complex seem to regulate Star-PAP activities and processivities toward RNA substrates, and unique sequence elements around the Star-PAP binding sites within the 3′-UTR of target genes contribute to Star-PAP specificity for processing. Therefore, Star-PAP and its regulatory molecules form a signaling nexus at the 3′-end of target mRNAs to control the expression of select group of genes including the ones involved in stress responses. PMID:23306079

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression

PubMed Central

Nahta, Rita; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Andrade-Vieira, Rafaela; Bay, Sarah; G. Brown, Dustin; Calaf, Gloria M.; Castellino, Robert C.; Cohen-Solal, Karine A.; Colacci, Annamaria; Cruickshanks, Nichola; Dent, Paul; Di Fiore, Riccardo; Forte, Stefano; Goldberg, Gary S.; Hamid, Roslida A.; Krishnan, Harini; Laird, Dale W.; Lasfar, Ahmed; Marignani, Paola A.; Memeo, Lorenzo; Mondello, Chiara; Naus, Christian C.; Ponce-Cusi, Richard; Raju, Jayadev; Roy, Debasish; Roy, Rabindra; P. Ryan, Elizabeth; Salem, Hosni K.; Scovassi, A. Ivana; Singh, Neetu; Vaccari, Monica; Vento, Renza; Vondráček, Jan; Wade, Mark; Woodrick, Jordan; Bisson, William H.

2015-01-01

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks. PMID:26106139

Pseudorabies virus-induced suppression of major histocompatibility complex class I antigen expression.

PubMed Central

Mellencamp, M W; O'Brien, P C; Stevenson, J R

1991-01-01

The ability of pseudorabies virus (PrV) to down-modulate expression of major histocompatibility complex class I antigens in murine and porcine cells was investigated. When quantified by flow cytometry, surface expression of class I Kk and Dk antigens on PrV-infected cells decreased by 60% or more. Down-modulation was associated with a decrease in total cellular class I antigens, indicating regulation at the transcriptional or posttranscriptional level. PrV did not suppress expression of transferrin receptor, suggesting a selective regulatory mechanism. Images PMID:1851884

Integration of cellular ubiquitin and membrane traffic systems: focus on deubiquitylases.

PubMed

Clague, Michael J; Urbé, Sylvie

2017-06-01

The cell is comprised of integrated multilevel protein networks or systems. The ubiquitin, protein homeostasis and membrane trafficking systems are highly integrated. Here, we look at the influence of reversible ubiquitylation on membrane trafficking and organelle dynamics. We review the regulation of endocytic sorting, selective autophagy and the secretory pathway by ubiquitin signals, with a particular focus on detailing the contribution of deubiquitylating enzymes. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Personalized regulation of glioblastoma cancer stem cells based on biomedical technologies: From theory to experiment (Review).

PubMed

Bryukhovetskiy, Igor; Ponomarenko, Arina; Lyakhova, Irina; Zaitsev, Sergey; Zayats, Yulia; Korneyko, Maria; Eliseikina, Marina; Mischenko, Polina; Shevchenko, Valerie; Shanker Sharma, Hari; Sharma, Aruna; Khotimchenko, Yuri

2018-08-01

Glioblastoma multiforme (GBM) is one of the most aggressive brain tumors. GBM represents >50% of primary tumors of the nervous system and ~20% of intracranial neoplasms. Standard treatment involves surgery, radiation and chemotherapy. However, the prognosis of GBM is usually poor, with a median survival of 15 months. Resistance of GBM to treatment can be explained by the presence of cancer stem cells (CSCs) among the GBM cell population. At present, there are no effective therapeutic strategies for the elimination of CSCs. The present review examined the nature of human GBM therapeutic resistance and attempted to systematize and put forward novel approaches for a personalized therapy of GBM that not only destroys tumor tissue, but also regulates cellular signaling and the morphogenetic properties of CSCs. The CSCs are considered to be an informationally accessible living system, and the CSC proteome should be used as a target for therapy directed at suppressing clonal selection mechanisms and CSC generation, destroying CSC hierarchy, and disrupting the interaction of CSCs with their microenvironment and extracellular matrix. These objectives can be achieved through the use of biomedical cellular products.

'Gardos Channelopathy': a variant of hereditary Stomatocytosis with complex molecular regulation.

PubMed

Fermo, Elisa; Bogdanova, Anna; Petkova-Kirova, Polina; Zaninoni, Anna; Marcello, Anna Paola; Makhro, Asya; Hänggi, Pascal; Hertz, Laura; Danielczok, Jens; Vercellati, Cristina; Mirra, Nadia; Zanella, Alberto; Cortelezzi, Agostino; Barcellini, Wilma; Kaestner, Lars; Bianchi, Paola

2017-05-11

The Gardos channel is a Ca 2+ sensitive, K + selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients' RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient's RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca 2+ overload in the RBCs of both patients and hypersensitivity of Ca 2+ uptake by RBCs to swelling. Altogether these findings suggest that the 'Gardos channelopathy' is a complex pathology, to some extent different from the common hereditary xerocytosis.

Metabolic gene regulation in a dynamically changing environment.

PubMed

Bennett, Matthew R; Pang, Wyming Lee; Ostroff, Natalie A; Baumgartner, Bridget L; Nayak, Sujata; Tsimring, Lev S; Hasty, Jeff

2008-08-28

Natural selection dictates that cells constantly adapt to dynamically changing environments in a context-dependent manner. Gene-regulatory networks often mediate the cellular response to perturbation, and an understanding of cellular adaptation will require experimental approaches aimed at subjecting cells to a dynamic environment that mimics their natural habitat. Here we monitor the response of Saccharomyces cerevisiae metabolic gene regulation to periodic changes in the external carbon source by using a microfluidic platform that allows precise, dynamic control over environmental conditions. We show that the metabolic system acts as a low-pass filter that reliably responds to a slowly changing environment, while effectively ignoring fast fluctuations. The sensitive low-frequency response was significantly faster than in predictions arising from our computational modelling, and this discrepancy was resolved by the discovery that two key galactose transcripts possess half-lives that depend on the carbon source. Finally, to explore how induction characteristics affect frequency response, we compare two S. cerevisiae strains and show that they have the same frequency response despite having markedly different induction properties. This suggests that although certain characteristics of the complex networks may differ when probed in a static environment, the system has been optimized for a robust response to a dynamically changing environment.

The frequencies of calcium oscillations are optimized for efficient calcium-mediated activation of Ras and the ERK/MAPK cascade.

PubMed

Kupzig, Sabine; Walker, Simon A; Cullen, Peter J

2005-05-24

Ras proteins are binary switches that, by cycling through inactive GDP- and active GTP-bound conformations, regulate multiple cellular signaling pathways, including those that control growth and differentiation. For some time, it has been known that receptor-mediated increases in the concentration of intracellular free calcium ([Ca(2+)](i)) can modulate Ras activation. Increases in [Ca(2+)](i) often occur as repetitive Ca(2+) spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca(2+) oscillations increase in frequency with the amplitude of receptor stimuli, a phenomenon critical for the induction of selective cellular functions. Here, we show that Ca(2+) oscillations are optimized for Ca(2+)-mediated activation of Ras and signaling through the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. We present additional evidence that Ca(2+) oscillations reduce the effective Ca(2+) threshold for the activation of Ras and that the oscillatory frequency is optimized for activation of Ras and the ERK/MAPK pathway. Our results describe a hitherto unrecognized link between complex Ca(2+) signals and the modulation of the Ras/ERK/MAPK signaling cascade.

Reciprocal Regulation of Endocytosis and Metabolism

PubMed Central

Antonescu, Costin N.; McGraw, Timothy E.; Klip, Amira

2014-01-01

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe3+, K+) or efflux (e.g., Na+) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis. PMID:24984778

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC

PubMed Central

Majumder, Mrinmoyee; House, Reniqua; Palanisamy, Nallasivam; Qie, Shuo; Day, Terrence A.; Neskey, David; Diehl, J. Alan

2016-01-01

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells. PMID:27606879

STATs shape the active enhancer landscape of T cell populations.

PubMed

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-Wei; Sartorelli, Vittorio; Kanno, Yuka; O'Shea, John J

2012-11-21

Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. Although enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4(+) T cells as a model of differentiation, mapping the activity of cell-type-specific enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. Copyright © 2012 Elsevier Inc. All rights reserved.

STATs Shape the Active Enhancer Landscape of T Cell Populations

PubMed Central

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-wei; Sartorelli, Vittorio; Kanno, Yuka; O’Shea, John J.

2012-01-01

SUMMARY Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. While enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4+ T cells as a model of differentiation, mapping the acquisition of cell-type-specific enhancer elements in T-helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the acquisition of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. PMID:23178119

Regulation of calcium channels in smooth muscle: New insights into the role of myosin light chain kinase

PubMed Central

Martinsen, A; Dessy, C; Morel, N

2014-01-01

Smooth muscle myosin light chain kinase (MLCK) plays a crucial role in artery contraction, which regulates blood pressure and blood flow distribution. In addition to this role, MLCK contributes to Ca2+ flux regulation in vascular smooth muscle (VSM) and in non-muscle cells, where cytoskeleton has been suggested to help Ca2+ channels trafficking. This conclusion is based on the use of pharmacological inhibitors of MLCK and molecular and cellular techniques developed to down-regulate the enzyme. Dissimilarities have been observed between cells and whole tissues, as well as between large conductance and small resistance arteries. A differential expression in MLCK and ion channels (either voltage-dependent Ca2+ channels or non-selective cationic channels) could account for these observations, and is in line with the functional properties of the arteries. A potential involvement of MLCK in the pathways modulating Ca2+ entry in VSM is described in the present review. PMID:25483583

Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells

PubMed Central

George, Laura A.; Acs, Andreas; Durrett, Russell E.

2018-01-01

Germinal centers (GCs) are the sites where B cells undergo affinity maturation. The regulation of cellular output from the GC is not well understood. Here, we show that from the earliest stages of the GC response, plasmablasts emerge at the GC–T zone interface (GTI). We define two main factors that regulate this process: Tfh-derived IL-21, which supports production of plasmablasts from the GC, and TNFSF13 (APRIL), which is produced by a population of podoplanin+ CD157high fibroblastic reticular cells located in the GTI that are also rich in message for IL-6 and chemokines CXCL12, CCL19, and CCL21. Plasmablasts in the GTI express the APRIL receptor TNFRSF13B (TACI), and blocking TACI interactions specifically reduces the numbers of plasmablasts appearing in the GTI. Plasma cells generated in the GTI may provide an early source of affinity-matured antibodies that may neutralize pathogens or provide feedback regulating GC B cell selection. PMID:29549115

Miklós Bodanszky Award Lecture: Advances in the selective targeting of protein phosphatase-1 and phosphatase-2A with peptides.

PubMed

Köhn, Maja

2017-10-01

Protein phosphatase-1 and phosphatase-2A are two ubiquitously expressed enzymes known to catalyze the majority of dephosphorylation reactions on serine and threonine inside cells. They play roles in most cellular processes and are tightly regulated by regulatory subunits in holoenzymes. Their misregulation and malfunction contribute to disease development and progression, such as in cancer, diabetes, viral infections, and neurological as well as heart diseases. Therefore, targeting these phosphatases for therapeutic use would be highly desirable; however, their complex regulation and high conservation of the active site have been major hurdles for selectively targeting them in the past. In the last decade, new approaches have been developed to overcome these hurdles and have strongly revived the field. I will focus here on peptide-based approaches, which contributed to showing that these phosphatases can be targeted selectively and aided in rethinking the design of selective phosphatase modulators. Finally, I will give a perspective on www.depod.org, the human dephosphorylation database, and how it can aid phosphatase modulator design. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Hippo Signaling: Key Emerging Pathway in Cellular and Whole-Body Metabolism.

PubMed

Ardestani, Amin; Lupse, Blaz; Maedler, Kathrin

2018-05-05

The evolutionarily conserved Hippo pathway is a key regulator of organ size and tissue homeostasis. Its dysregulation is linked to multiple pathological disorders. In addition to regulating development and growth, recent studies show that Hippo pathway components such as MST1/2 and LATS1/2 kinases, as well as YAP/TAZ transcriptional coactivators, are regulated by metabolic pathways and that the Hippo pathway controls metabolic processes at the cellular and organismal levels in physiological and metabolic disease states such as obesity, type 2 diabetes (T2D), nonalcoholic fatty liver disease (NAFLD), cardiovascular disorders, and cancer. In this review we summarize the connection between key Hippo components and metabolism, and how this interplay regulates cellular metabolism and metabolic pathways. The emerging function of Hippo in the regulation of metabolic homeostasis under physiological and pathological conditions is highlighted. Copyright © 2018 Elsevier Ltd. All rights reserved.

O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway.

PubMed

Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen

2016-03-18

O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. Copyright © 2016 Elsevier Inc. All rights reserved.

Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

PubMed Central

Ammer, Amanda Gatesman; Weed, Scott A.

2008-01-01

Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630

AMP-Activated Protein Kinase – A Ubiquitous Signalling Pathway with Key Roles in the Cardiovascular System

PubMed Central

Salt, Ian P.; Hardie, D. Grahame

2017-01-01

The AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole body energy homeostasis, which acts to restore energy homoeostasis whenever cellular energy charge is depleted. Over the last two decades, it has become apparent that AMPK regulates a number of other cellular functions and has specific roles in cardiovascular tissues, acting to regulate cardiac metabolism and contractile function as well as promoting anti-contractile, anti-inflammatory and anti-atherogenic actions in blood vessels. In this review, we will discuss the role of AMPK in the cardiovascular system, including the molecular basis of mutations in AMPK that alter cardiac physiology and the proposed mechanisms by which AMPK regulates vascular function under physiological and pathophysiological conditions. PMID:28546359

Interface Pattern Selection in Directional Solidification

NASA Technical Reports Server (NTRS)

Trivedi, Rohit; Tewari, Surendra N.

2001-01-01

The central focus of this research is to establish key scientific concepts that govern the selection of cellular and dendritic patterns during the directional solidification of alloys. Ground-based studies have established that the conditions under which cellular and dendritic microstructures form are precisely where convection effects are dominant in bulk samples. Thus, experimental data can not be obtained terrestrially under pure diffusive regime. Furthermore, reliable theoretical models are not yet possible which can quantitatively incorporate fluid flow in the pattern selection criterion. Consequently, microgravity experiments on cellular and dendritic growth are designed to obtain benchmark data under diffusive growth conditions that can be quantitatively analyzed and compared with the rigorous theoretical model to establish the fundamental principles that govern the selection of specific microstructure and its length scales. In the cellular structure, different cells in an array are strongly coupled so that the cellular pattern evolution is controlled by complex interactions between thermal diffusion, solute diffusion and interface effects. These interactions give infinity of solutions, and the system selects only a narrow band of solutions. The aim of this investigation is to obtain benchmark data and develop a rigorous theoretical model that will allow us to quantitatively establish the physics of this selection process.

KDM1 Class Flavin-Dependent Protein Lysine Demethylases

PubMed Central

Burg, Jonathan M.; Link, Jennifer E.; Morgan, Brittany S.; Heller, Frederick J.; Hargrove, Amanda E.; McCafferty, Dewey G.

2015-01-01

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1 -selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance. PMID:25787087

Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight

DOE Office of Scientific and Technical Information (OSTI.GOV)

Touré, B. Barry; Giraldes, John; Smith, Troy

2016-05-26

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency.more » These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.« less

Socializing with MYC: cell competition in development and as a model for premalignant cancer.

PubMed

Johnston, Laura A

2014-04-01

Studies in Drosophila and mammals have made it clear that genetic mutations that arise in somatic tissues are rapidly recognized and eliminated, suggesting that cellular fitness is tightly monitored. During development, damaged, mutant, or otherwise unfit cells are prevented from contributing to the tissue and are instructed to die, whereas healthy cells benefit and populate the animal. This cell selection process, known as cell competition, eliminates somatic genetic heterogeneity and promotes tissue fitness during development. Yet cell competition also has a dark side. Super competition can be exploited by incipient cancers to subvert cellular cooperation and promote selfish behavior. Evidence is accumulating that MYC plays a key role in regulation of social behavior within tissues. Given the high number of tumors with deregulated MYC, studies of cell competition promise to yield insight into how the local environment yields to and participates in the early stages of tumor formation.

Genetic incorporation of Nε-acetyllysine reveals a novel acetylation-sumoylation switch in yeast.

PubMed

Kim, Sang-Woo; Lee, Kyung Jin; Kim, Sinil; Kim, Jihyo; Cho, Kyukwang; Ro, Hyeon-Su; Park, Hee-Sung

2017-11-01

The lysine acetylation of proteins plays a key role in regulating protein functions, thereby controlling a wide range of cellular processes. Despite the prevalence and significance of lysine acetylation in eukaryotes, however, its systematic study has been challenged by the technical limitations of conventional approaches for selective lysine acetylation in vivo. Here, we report the in vivo study of lysine acetylation via the genetic incorporation of N ε -acetyllysine in yeast. We demonstrate that a newly discovered acetylation-sumoylation switch precisely controls the localization and cellular function of the yeast septin protein, Cdc11, during the cell cycle. This approach should facilitate the comprehensive in vivo study of lysine acetylation across a wide range of proteins in eukaryotic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

PINK1 deficiency enhances autophagy and mitophagy induction.

PubMed

Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Martin-Cano, Francisco E; González-Soltero, María E; Tandon, Anurag; Fuentes, José M; González-Polo, Rosa A

2016-03-01

Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control.

PINK1 deficiency enhances autophagy and mitophagy induction

PubMed Central

Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Martin-Cano, Francisco E; González-Soltero, María E; Tandon, Anurag; Fuentes, José M; González-Polo, Rosa A

2016-01-01

Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control. PMID:27308585

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

PubMed Central

Billaud, Marie; Lohman, Alexander W.; Johnstone, Scott R.; Biwer, Lauren A.; Mutchler, Stephanie; Isakson, Brant E.

2014-01-01

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function. PMID:24671377

Regulation of cellular communication by signaling microdomains in the blood vessel wall.

PubMed

Billaud, Marie; Lohman, Alexander W; Johnstone, Scott R; Biwer, Lauren A; Mutchler, Stephanie; Isakson, Brant E

2014-01-01

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function.

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells.

PubMed

Li, Lingyun; Steinauer, Kirsten K; Dirks, Amie J; Husbeck, Bryan; Gibbs, Iris; Knox, Susan J

2003-12-01

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

Are apparent negative effects of feeding GM MON810 maize to Atlantic salmon, Salmo salar, caused by confounding factors?

PubMed

Sissener, Nini H; Hemre, Gro-Ingunn; Lall, Santosh P; Sagstad, Anita; Petersen, Kjell; Williams, Jason; Rohloff, Jens; Sanden, Monica

2011-07-01

The present study was conducted to follow up on apparent differences in growth, relative organ sizes, cellular stress and immune function in Atlantic salmon fed feed containing GM Bacillus thuringiensis maize compared with feed containing the non-modified parental maize line. Gene expression profiling on the distal intestinal segment and liver was performed by microarray, and selected genes were followed up by quantitative PCR (qPCR). In the liver, qPCR revealed some differentially regulated genes, including up-regulation of gelsolin precursor, down-regulation of ferritin heavy subunit and a tendency towards down-regulation of metallothionein (MT)-B. This, combined with the up-regulation of anti-apoptotic protein NR13 and similar tendencies for ferritin heavy chain and MT-A and -B in the distal intestine, suggests changes in cellular stress/antioxidant status. This corresponds well with and strengthens previous findings in these fish. To exclude possible confounding factors, the maize ingredients were analysed for mycotoxins and metabolites. The GM maize contained 90 μg/kg of deoxynivalenol (DON), while the non-GM maize was below the detection limit. Differences were also observed in the metabolite profiles of the two maize varieties, some of which seemed connected to the mycotoxin level. The effects on salmon observed in the present and previous studies correspond relatively well with the effects of DON as reported in the literature for other production animals, but knowledge regarding effects and harmful dose levels in fish is scarce. Thus, it is difficult to conclude whether the observed effects are caused by the DON level or by some other aspect of the GM maize ingredient.

Identification of miRNAs during mouse postnatal ovarian development and superovulation.

PubMed

Khan, Hamid Ali; Zhao, Yi; Wang, Li; Li, Qian; Du, Yu-Ai; Dan, Yi; Huo, Li-Jun

2015-07-08

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16-26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

A multi-model approach to nucleic acid-based drug development.

PubMed

Gautherot, Isabelle; Sodoyer, Regís

2004-01-01

With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies. Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency. Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic. Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product. Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites. Individually, each strategy has its drawbacks from a drug development perspective. Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment. In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis. Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications. Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response. For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such approaches also have limitations. To streamline nucleic acid drug discovery, we propose a multi-model strategy integrating high-throughput-adapted bacterial screening, followed by reporter-based and/or natural cellular models and potentially also in vitro assays for characterization of the most promising candidate sequences, before final in vivo testing.

Increased Toxicity of Karenia brevis during Phosphate Limited Growth: Ecological and Evolutionary Implications

PubMed Central

Hardison, Donnie Ransom; Sunda, William G.; Shea, Damian; Litaker, Richard Wayne

2013-01-01

Karenia brevis is the dominant toxic red tide algal species in the Gulf of Mexico. It produces potent neurotoxins (brevetoxins [PbTxs]), which negatively impact human and animal health, local economies, and ecosystem function. Field measurements have shown that cellular brevetoxin contents vary from 1–68 pg/cell but the source of this variability is uncertain. Increases in cellular toxicity caused by nutrient-limitation and inter-strain differences have been observed in many algal species. This study examined the effect of P-limitation of growth rate on cellular toxin concentrations in five Karenia brevis strains from different geographic locations. Phosphorous was selected because of evidence for regional P-limitation of algal growth in the Gulf of Mexico. Depending on the isolate, P-limited cells had 2.3- to 7.3-fold higher PbTx per cell than P-replete cells. The percent of cellular carbon associated with brevetoxins (%C-PbTx) was ∼ 0.7 to 2.1% in P-replete cells, but increased to 1.6–5% under P-limitation. Because PbTxs are potent anti-grazing compounds, this increased investment in PbTxs should enhance cellular survival during periods of nutrient-limited growth. The %C-PbTx was inversely related to the specific growth rate in both the nutrient-replete and P-limited cultures of all strains. This inverse relationship is consistent with an evolutionary tradeoff between carbon investment in PbTxs and other grazing defenses, and C investment in growth and reproduction. In aquatic environments where nutrient supply and grazing pressure often vary on different temporal and spatial scales, this tradeoff would be selectively advantageous as it would result in increased net population growth rates. The variation in PbTx/cell values observed in this study can account for the range of values observed in the field, including the highest values, which are not observed under N-limitation. These results suggest P-limitation is an important factor regulating cellular toxicity and adverse impacts during at least some K. brevis blooms. PMID:23554901

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

PubMed Central

Choi, Kuicheon; Mollapour, Elahe; Choi, Jae H.; Shears, Stephen B.

2009-01-01

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP]2-InsP4 synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP]2-InsP4 synthesis without affecting other inositol phosphates. In addition, we report that the short-term increases in [PP]2-InsP4 levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP]2-InsP4 synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2′-amino-3′-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP]2-InsP4 is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks. PMID:18460607

Targeting ligand-operated chaperone sigma-1 receptors in the treatment of neuropsychiatric disorders

PubMed Central

Teruo, Hayashi; Shang-Yi, Tsai; Tomohisa, Mori; Michiko, Fujimoto; Tsung-Ping, Su

2011-01-01

Introduction Current conventional therapeutic drugs for the treatment of psychiatric or neurodegenerative disorders have certain limitations of use. Psychotherapeutic drugs such as typical and atypical antipsychotics, tricyclic antidepressants, and selective monoamine reuptake inhibitors, aim to normalize the hyper- or hypo-neurotransmission of monoaminergic systems. Despite their great contribution to the outcomes of psychiatric patients, these agents often exert severe side effects and require chronic treatments to promote amelioration of symptoms. Furthermore, drugs available for the treatment of neurodegenerative disorders are severely limited. Areas covered This review discusses recent evidence that has shed light on sigma-1 receptor ligands, which may serve as a new class of antidepressants or neuroprotective agents. Sigma-1 receptors are novel ligand-operated molecular chaperones regulating a variety of signal transduction, ER stress, cellular redox, cellular survival, and synaptogenesis. Selective sigma-1 receptor ligands exert rapid antidepressant-like, anxiolytic, antinociceptive and robust neuroprotective actions in preclinical studies. The review also looks at recent studies which suggest that reactive oxygen species might play a crucial role as signal integrators at the downstream of Sig-1Rs Expert opinion The significant advances in sigma receptor research in the last decade have begun to elucidate the intracellular signal cascades upstream and downstream of sigma-1 receptors. The novel ligand-operated properties of the sigma-1 receptor chaperone may enable a variety of interventions by which stress-related cellular systems are pharmacologically controlled. PMID:21375464

Cellular volume regulation and substrate stiffness modulate the detachment dynamics of adherent cells

NASA Astrophysics Data System (ADS)

Yang, Yuehua; Jiang, Hongyuan

2018-03-01

Quantitative characterizations of cell detachment are vital for understanding the fundamental mechanisms of cell adhesion. Experiments have found that cell detachment shows strong rate dependence, which is mostly attributed to the binding-unbinding kinetics of receptor-ligand bond. However, our recent study showed that the cellular volume regulation can significantly regulate the dynamics of adherent cell and cell detachment. How this cellular volume regulation contributes to the rate dependence of cell detachment remains elusive. Here, we systematically study the role of cellular volume regulation in the rate dependence of cell detachment by investigating the cell detachments of nonspecific adhesion and specific adhesion. We find that the cellular volume regulation and the bond kinetics dominate the rate dependence of cell detachment at different time scales. We further test the validity of the traditional Johnson-Kendall-Roberts (JKR) contact model and the detachment model developed by Wyart and Gennes et al (W-G model). When the cell volume is changeable, the JKR model is not appropriate for both the detachments of convex cells and concave cells. The W-G model is valid for the detachment of convex cells but is no longer applicable for the detachment of concave cells. Finally, we show that the rupture force of adherent cells is also highly sensitive to substrate stiffness, since an increase in substrate stiffness will lead to more associated bonds. These findings can provide insight into the critical role of cell volume in cell detachment and might have profound implications for other adhesion-related physiological processes.

Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation

PubMed Central

Niessen, Carien M.; Leckband, Deborah; Yap, Alpha S.

2013-01-01

This review addresses the cellular and molecular mechanisms of cadherin-based tissue morphogenesis. Tissue physiology is profoundly influenced by the distinctive organizations of cells in organs and tissues. In metazoa, adhesion receptors of the classical cadherin family play important roles in establishing and maintaining such tissue organization. Indeed, it is apparent that cadherins participate in a range of morphogenetic events that range from support of tissue integrity to dynamic cellular rearrangements. A comprehensive understanding of cadherin-based morphogenesis must then define the molecular and cellular mechanisms that support these distinct cadherin biologies. Here we focus on four key mechanistic elements: the molecular basis for adhesion through cadherin ectodomains; the regulation of cadherin expression at the cell surface; cooperation between cadherins and the actin cytoskeleton; and regulation by cell signaling. We discuss current progress and outline issues for further research in these fields. PMID:21527735

Toward a systems-level view of dynamic phosphorylation networks

PubMed Central

Newman, Robert H.; Zhang, Jin; Zhu, Heng

2014-01-01

To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks. PMID:25177341

EZH2 mediates lidamycin-induced cellular senescence through regulating p21 expression in human colon cancer cells

PubMed Central

Sha, Ming-Quan; Zhao, Xiao-Li; Li, Liang; Li, Li-Hui; Li, Yi; Dong, Tian-Geng; Niu, Wei-Xin; Jia, Li-Jun; Shao, Rong-Guang; Zhen, Yong-Su; Wang, Zhen

2016-01-01

Lidamycin (LDM) is a novel member of the enediyne antibiotics identified in China with potent antitumor activity. However, it remains unclear whether LDM has potential molecular targets that may affect its antitumor activity. Enhancer of zeste homolog 2 (EZH2) functions as a histone lysine methyltransferase and mediates trimethylation on histone 3 lysine 27 (H3K27me3). High EZH2 level is found to be positively correlated with the aggressiveness, metastasis and poor prognosis of cancer. Here, we aim to study the role of EZH2 in LDM-induced senescence, as well as in the cytotoxicity of LDM in human colon cancer cells. LDM is found to be relatively more potent in inhibiting the colon cancer cells harboring high EZH2 level and induces irreversible cellular senescence at IC50 dose range, as evidenced by senescence-associated β-galactosidase staining, cell cycle arrest and molecular changes of senescence regulators including p21 in HCT116 and SW620 cells. More importantly, LDM is found to markedly inhibit EZH2 expression at both protein and mRNA levels upon the induction of p21 and cellular senescence. LDM also selectively inhibits EZH2 expression as compared with other histone lysine methyltransferases. Knockdown of p21 with siRNAs abolishes LDM-induced senescence, whereas EZH2 knockdown markedly increases p21 expression and causes senescent phenotype. Enrichment of both EZH2 and H3K27me3 levels in the p21 promoter region is reduced by LDM. Moreover, EZH2 overexpression reduces cellular senescence, p21 expression and DNA damage response upon LDM exposure. LDM also demonstrates potent antitumor efficacy in xenografted animal models. Collectively, our work provides first demonstration that EZH2 may mediate, at least partially, the senescence-inducing effects of LDM by regulating p21 expression and DNA damage effect. Thus, EZH2 may serve as a potential target and biomarker to indicate the clinical efficacy of the potent enediyne antitumor drug. PMID:27882937

AGCVIII Kinases: at the crossroads of cellular signaling

USDA-ARS?s Scientific Manuscript database

AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

Epstein-Barr virus growth/latency III program alters cellular microRNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cameron, Jennifer E.; Tulane Cancer Center, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL79, New Orleans, LA 70112; Fewell, Claire

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lowermore » in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.« less

Structural Implications for Selective Targeting of PARPs.

PubMed

Steffen, Jamin D; Brody, Jonathan R; Armen, Roger S; Pascal, John M

2013-12-20

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD(+) as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed "synthetic lethality." In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients.

Structural Implications for Selective Targeting of PARPs

PubMed Central

Steffen, Jamin D.; Brody, Jonathan R.; Armen, Roger S.; Pascal, John M.

2013-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD+ as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed “synthetic lethality.” In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients. PMID:24392349

Cellular and molecular mechanisms regulating vascular tone. Part 1: basic mechanisms controlling cytosolic Ca2+ concentration and the Ca2+-dependent regulation of vascular tone.

PubMed

Akata, Takashi

2007-01-01

General anesthetics cause hemodynamic instability and alter blood flow to various organs. There is mounting evidence that most general anesthetics, at clinical concentrations, influence a wide variety of cellular and molecular mechanisms regulating the contractile state of vascular smooth muscle cells (i.e., vascular tone). In addition, in current anesthetic practice, various types of vasoactive agents are often used to control vascular reactivity and to sustain tissue blood flow in high-risk surgical patients with impaired vital organ function and/or hemodynamic instability. Understanding the physiological mechanisms involved in the regulation of vascular tone thus would be beneficial for anesthesiologists. This review, in two parts, provides an overview of current knowledge about the cellular and molecular mechanisms regulating vascular tone-i.e., targets for general anesthetics, as well as for vasoactive drugs that are used in intraoperative circulatory management. This first part of the two-part review focuses on basic mechanisms regulating cytosolic Ca2+ concentration and the Ca2+-dependent regulation of vascular tone.

Piezo proteins: regulators of mechanosensation and other cellular processes.

PubMed

Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

2014-11-14

Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic Feedback Regulation of Frontal Cortical Neuronal Ensembles Through Activity-Dependent Arc Expression and Dopaminergic Input.

PubMed

Mastwal, Surjeet; Cao, Vania; Wang, Kuan Hong

2016-01-01

Mental functions involve coordinated activities of specific neuronal ensembles that are embedded in complex brain circuits. Aberrant neuronal ensemble dynamics is thought to form the neurobiological basis of mental disorders. A major challenge in mental health research is to identify these cellular ensembles and determine what molecular mechanisms constrain their emergence and consolidation during development and learning. Here, we provide a perspective based on recent studies that use activity-dependent gene Arc/Arg3.1 as a cellular marker to identify neuronal ensembles and a molecular probe to modulate circuit functions. These studies have demonstrated that the transcription of Arc is activated in selective groups of frontal cortical neurons in response to specific behavioral tasks. Arc expression regulates the persistent firing of individual neurons and predicts the consolidation of neuronal ensembles during repeated learning. Therefore, the Arc pathway represents a prototypical example of activity-dependent genetic feedback regulation of neuronal ensembles. The activation of this pathway in the frontal cortex starts during early postnatal development and requires dopaminergic (DA) input. Conversely, genetic disruption of Arc leads to a hypoactive mesofrontal dopamine circuit and its related cognitive deficit. This mutual interaction suggests an auto-regulatory mechanism to amplify the impact of neuromodulators and activity-regulated genes during postnatal development. Such a mechanism may contribute to the association of mutations in dopamine and Arc pathways with neurodevelopmental psychiatric disorders. As the mesofrontal dopamine circuit shows extensive activity-dependent developmental plasticity, activity-guided modulation of DA projections or Arc ensembles during development may help to repair circuit deficits related to neuropsychiatric disorders.

Rheb may complex with RASSF1A to coordinate Hippo and TOR signaling.

PubMed

Nelson, Nicholas; Clark, Geoffrey J

2016-06-07

The TOR pathway is a vital component of cellular homeostasis that controls the synthesis of proteins, nucleic acids and lipids. Its core is the TOR kinase. Activation of the TOR pathway suppresses autophagy, which plays a vital but complex role in tumorigenesis. The TOR pathway is regulated by activation of the Ras-related protein Rheb, which can bind mTOR. The Hippo pathway is a major growth control module that regulates cell growth, differentiation and apoptosis. Its core consists of an MST/LATS kinase cascade that can be activated by the RASSF1A tumor suppressor. The TOR and Hippo pathways may be coordinately regulated to promote cellular homeostasis. However, the links between the pathways remain only partially understood. We now demonstrate that in addition to mTOR regulation, Rheb also impacts the Hippo pathway by forming a complex with RASSF1A. Using stable clones of two human lung tumor cell lines (NCI-H1792 and NCI-H1299) with shRNA-mediated silencing or ectopic overexpression of RASSF1A, we show that activated Rheb stimulates the Hippo pathway, but is suppressed in its ability to stimulate the TOR pathway. Moreover, by selectively labeling autophagic vacuoles we show that RASSF1A inhibits the ability of Rheb to suppress autophagy and enhance cell growth. Thus, we identify a new connection that impacts coordination of Hippo and TOR signaling. As RASSF1A expression is frequently lost in human tumors, the RASSF1A status of a tumor may impact not just its Hippo pathway status, but also its TOR pathway status.

Circadian transcription factor BMAL1 regulates innate immunity against select RNA viruses.

PubMed

Majumdar, Tanmay; Dhar, Jayeeta; Patel, Sonal; Kondratov, Roman; Barik, Sailen

2017-02-01

BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3 or ARNT3) belongs to the family of the basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, and is a key component of the molecular oscillator that generates circadian rhythms. Here, we report that BMAL1-deficient cells are significantly more susceptible to infection by two major respiratory viruses of the Paramyxoviridae family, namely RSV and PIV3. Embryonic fibroblasts from Bmal1 -/- mice produced nearly 10-fold more progeny virus than their wild type controls. These results were supported by animal studies whereby pulmonary infection of RSV produced a more severe disease and morbidity in Bmal1 -/- mice. These results show that BMAL1 can regulate cellular innate immunity against specific RNA viruses.

Current perspectives on the use of ancillary materials for the manufacture of cellular therapies.

PubMed

Solomon, Jennifer; Csontos, Lynn; Clarke, Dominic; Bonyhadi, Mark; Zylberberg, Claudia; McNiece, Ian; Kurtzberg, Joanne; Bell, Rosemarie; Deans, Robert

2016-01-01

Continued growth in the cell therapy industry and commercialization of cell therapies that successfully advance through clinical trials has led to increased awareness around the need for specialized and complex materials utilized in their manufacture. Ancillary materials (AMs) are components or reagents used during the manufacture of cell therapy products but are not intended to be part of the final products. Commonly, there are limitations in the availability of clinical-grade reagents used as AMs. Furthermore, AMs may affect the efficacy of the cell product and subsequent safety of the cell therapy for the patient. As such, AMs must be carefully selected and appropriately qualified during the cell therapy development process. However, the ongoing evolution of cell therapy research, limited number of clinical trials and registered cell therapy products results in the current absence of specific regulations governing the composition, compliance, and qualification of AMs often leads to confusion by suppliers and users in this field. Here we provide an overview and interpretation of the existing global framework surrounding AM use and investigate some common misunderstandings within the industry, with the aim of facilitating the appropriate selection and qualification of AMs. The key message we wish to emphasize is that in order to most effectively mitigate risk around cell therapy development and patient safety, users must work with their suppliers and regulators to qualify each AM to assess source, purity, identity, safety, and suitability in a given application. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

PubMed

Matalon, Omri; Ben-Shmuel, Aviad; Kivelevitz, Jessica; Sabag, Batel; Fried, Sophia; Joseph, Noah; Noy, Elad; Biber, Guy; Barda-Saad, Mira

2018-03-01

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems. © 2018 The Authors.

Vaccine induced differential expressions of miRNAs at cytolytic stage in chickens resistant or susceptible to Marek’s disease

USDA-ARS?s Scientific Manuscript database

Gene expression regulation is critical for all cellular processes since dysregulation of it often results in elevated disease risk and compromised cellular immunity. MicroRNAs (miRNAs) directly regulate gene expression post-transcriptionally through base-pairing with regions in the 3’-untranslated s...

Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes

PubMed Central

Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram

2012-01-01

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes. PMID:22684502

IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Handayaningsih, Anastasia-Evi; Takahashi, Michiko; Fukuoka, Hidenori

2012-08-24

Highlights: Black-Right-Pointing-Pointer Cellular senescence plays an important role in tumorigenesis and aging process. Black-Right-Pointing-Pointer We demonstrated IGF-I enhanced cellular senescence in primary confluent cells. Black-Right-Pointing-Pointer IGF-I enhanced cellular senescence in the ROS and p53-dependent manner. Black-Right-Pointing-Pointer These results may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging. -- Abstract: Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated {beta}-galactosidase (SA-{beta}-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the presentmore » study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, {gamma}H2AX, the increased levels of p53 and p21 proteins, and activated SA-{beta}-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-{beta}-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging.« less

Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells.

PubMed

Chang, Elizabeth T; Parekh, Palak R; Yang, Qingyuan; Nguyen, Duc M; Carrier, France

2016-03-01

The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3'UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells.

Cellular basis of morphological variation and temperature-related plasticity in Drosophila melanogaster strains with divergent wing shapes.

PubMed

Torquato, Libéria Souza; Mattos, Daniel; Matta, Bruna Palma; Bitner-Mathé, Blanche Christine

2014-12-01

Organ shape evolves through cross-generational changes in developmental patterns at cellular and/or tissue levels that ultimately alter tissue dimensions and final adult proportions. Here, we investigated the cellular basis of an artificially selected divergence in the outline shape of Drosophila melanogaster wings, by comparing flies with elongated or rounded wing shapes but with remarkably similar wing sizes. We also tested whether cellular plasticity in response to developmental temperature was altered by such selection. Results show that variation in cellular traits is associated with wing shape differences, and that cell number may play an important role in wing shape response to selection. Regarding the effects of developmental temperature, a size-related plastic response was observed, in that flies reared at 16 °C developed larger wings with larger and more numerous cells across all intervein regions relative to flies reared at 25 °C. Nevertheless, no conclusive indication of altered phenotypic plasticity was found between selection strains for any wing or cellular trait. We also described how cell area is distributed across different intervein regions. It follows that cell area tends to decrease along the anterior wing compartment and increase along the posterior one. Remarkably, such pattern was observed not only in the selected strains but also in the natural baseline population, suggesting that it might be canalized during development and was not altered by the intense program of artificial selection for divergent wing shapes.

Regulation of Plant Cellular and Organismal Development by SUMO.

PubMed

Elrouby, Nabil

2017-01-01

This chapter clearly demonstrates the breadth and spectrum of the processes that SUMO regulates during plant development. The gross phenotypes observed in mutants of the SUMO conjugation and deconjugation enzymes reflect these essential roles, and detailed analyses of these mutants under different growth conditions revealed roles in biotic and abiotic stress responses, phosphate starvation, nitrate and sulphur metabolism, freezing and drought tolerance and response to excess copper. SUMO functions also intersect with those regulated by several hormones such as salicylic acid , abscisic acid , gibberellins and auxin, and detailed studies provide mechanistic clues of how sumoylation may regulate these processes. The regulation of COP1 and PhyB functions by sumoylation provides very strong evidence that SUMO is heavily involved in the regulation of light signaling in plants. At the cellular and subcellular levels, SUMO regulates meristem architecture, the switch from the mitotic cycle into the endocycle, meiosis, centromere decondensation and exit from mitosis, transcriptional control, and release from transcriptional silencing. Most of these advances in our understanding of SUMO functions during plant development emerged over the past 6-7 years, and they may only predict a prominent rise of SUMO as a major regulator of eukaryotic cellular and organismal growth and development.

Cellular Response to Ionizing Radiation: A MicroRNA Story

PubMed Central

Halimi, Mohammad; Asghari, S. Mohsen; Sariri, Reyhaneh; Moslemi, Dariush; Parsian, Hadi

2012-01-01

MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play a crucial role in diverse cellular pathways. Ionizing radiation (IR) is one of the most important treatment protocols for patients that suffer from cancer and affects directly or indirectly cellular integration. Recently it has been discovered that microRNA-mediated gene regulation interferes with radio-related pathways in ionizing radiation. Here, we review the recent discoveries about miRNAs in cellular response to IR. Thoroughly understanding the mechanism of miRNAs in radiation response, it will be possible to design new strategies for improving radiotherapy efficiency and ultimately cancer treatment. PMID:24551775

Sub-cellular distribution and translocation of TRP channels.

PubMed

Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

2011-01-01

Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

RNA fluorescence with light-up aptamers

NASA Astrophysics Data System (ADS)

Ouellet, Jonathan

2016-06-01

Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way.

Integrated regulation of PIKK-mediated stress responses by AAA+ proteins RUVBL1 and RUVBL2

PubMed Central

Izumi, Natsuko; Yamashita, Akio; Ohno, Shigeo

2012-01-01

Proteins of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family are activated by various cellular stresses, including DNA damage, premature termination codon and nutritional status, and induce appropriate cellular responses. The importance of PIKK functions in the maintenance of genome integrity, accurate gene expression and the proper control of cell growth/proliferation is established. Recently, ATPase associated diverse cellular activities (AAA+) proteins RUVBL1 and RUVBL2 (RUVBL1/2) have been shown to be common regulators of PIKKs. The RUVBL1/2 complex regulates PIKK-mediated stress responses through physical interactions with PIKKs and by controlling PIKK mRNA levels. In this review, the functions of PIKKs in stress responses are outlined and the physiological significance of the integrated regulation of PIKKs by the RUVBL1/2 complex is presented. We also discuss a putative “PIKK regulatory chaperone complex” including other PIKK regulators, Hsp90 and the Tel2 complex. PMID:22540023

Redox-dependent transcriptional regulation.

PubMed

Liu, Hongjun; Colavitti, Renata; Rovira, Ilsa I; Finkel, Toren

2005-11-11

Reactive oxygen species contribute to the pathogenesis of a number of disparate disorders including tissue inflammation, heart failure, hypertension, and atherosclerosis. In response to oxidative stress, cells activate expression of a number of genes, including those required for the detoxification of reactive molecules as well as for the repair and maintenance of cellular homeostasis. In many cases, these induced genes are regulated by transcription factors whose structure, subcellular localization, or affinity for DNA is directly or indirectly regulated by the level of oxidative stress. This review summarizes the recent progress on how cellular redox status can regulate transcription-factor activity and the implications of this regulation for cardiovascular disease.

A novel strategy for global mapping of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry identification.

PubMed

Shen, Bingquan; Zhang, Wanjun; Shi, Zhaomei; Tian, Fang; Deng, Yulin; Sun, Changqing; Wang, Guangshun; Qin, Weijie; Qian, Xiaohong

2017-07-01

O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine. Copyright © 2017. Published by Elsevier B.V.

Exploring the Genetic Signature of Body Size in Yucatan Miniature Pig

PubMed Central

Kim, Hyeongmin; Song, Ki Duk; Kim, Hyeon Jeong; Park, WonCheoul; Kim, Jaemin; Lee, Taeheon; Shin, Dong-Hyun; Kwak, Woori; Kwon, Young-jun; Sung, Samsun; Moon, Sunjin; Lee, Kyung-Tai; Kim, Namshin; Hong, Joon Ki; Eo, Kyung Yeon; Seo, Kang Seok; Kim, Girak; Park, Sungmoo; Yun, Cheol-Heui; Kim, Hyunil; Choi, Kimyung; Kim, Jiho; Lee, Woon Kyu; Kim, Duk-Kyung; Oh, Jae-Don; Kim, Eui-Soo; Cho, Seoae; Lee, Hak-Kyo; Kim, Tae-Hun; Kim, Heebal

2015-01-01

Since being domesticated about 10,000–12,000 years ago, domestic pigs (Sus scrofa domesticus) have been selected for traits of economic importance, in particular large body size. However, Yucatan miniature pigs have been selected for small body size to withstand high temperature environment and for laboratory use. This renders the Yucatan miniature pig a valuable model for understanding the evolution of body size. We investigate the genetic signature for selection of body size in the Yucatan miniature pig. Phylogenetic distance of Yucatan miniature pig was compared to other large swine breeds (Yorkshire, Landrace, Duroc and wild boar). By estimating the XP-EHH statistic using re-sequencing data derived from 70 pigs, we were able to unravel the signatures of selection of body size. We found that both selections at the level of organism, and at the cellular level have occurred. Selection at the higher levels include feed intake, regulation of body weight and increase in mass while selection at the molecular level includes cell cycle and cell proliferation. Positively selected genes probed by XP-EHH may provide insight into the docile character and innate immunity as well as body size of Yucatan miniature pig. PMID:25885114

Cardiac system bioenergetics: metabolic basis of the Frank-Starling law

PubMed Central

Saks, Valdur; Dzeja, Petras; Schlattner, Uwe; Vendelin, Marko; Terzic, Andre; Wallimann, Theo

2006-01-01

The fundamental principle of cardiac behaviour is described by the Frank-Starling law relating force of contraction during systole with end-diastolic volume. While both work and respiration rates increase linearly with imposed load, the basis of mechano-energetic coupling in heart muscle has remained a long-standing enigma. Here, we highlight advances made in understanding of complex cellular and molecular mechanisms that orchestrate coupling of mitochondrial oxidative phosphorylation with ATP utilization for muscle contraction. Cardiac system bioenergetics critically depends on an interrelated metabolic infrastructure regulating mitochondrial respiration and energy fluxes throughout cellular compartments. The data reviewed indicate the significance of two interrelated systems regulating mitochondrial respiration and energy fluxes in cells: (1) the creatine kinase, adenylate kinase and glycolytic pathways that communicate flux changes generated by cellular ATPases within structurally organized enzymatic modules and networks; and (2) a secondary system based on mitochondrial participation in cellular calcium cycle, which adjusts substrate oxidation and energy-transducing processes to meet increasing cellular energy demands. By conveying energetic signals to metabolic sensors, coupled phosphotransfer reactions provide a high-fidelity regulation of the excitation–contraction cycle. Such integration of energetics with calcium signalling systems provides the basis for ‘metabolic pacing’, synchronizing the cellular electrical and mechanical activities with energy supply processes. PMID:16410283

Cell Cycle Regulators Guide Mitochondrial Activity in Radiation-Induced Adaptive Response

PubMed Central

Alexandrou, Aris T.

2014-01-01

Abstract Significance: There are accruing concerns on potential genotoxic agents present in the environment including low-dose ionizing radiation (LDIR) that naturally exists on earth's surface and atmosphere and is frequently used in medical diagnosis and nuclear industry. Although its long-term health risk is being evaluated and remains controversial, LDIR is shown to induce temporary but significant adaptive responses in mammalian cells and animals. The mechanisms guiding the mitochondrial function in LDIR-induced adaptive response represent a unique communication between DNA damage and cellular metabolism. Elucidation of the LDIR-regulated mitochondrial activity may reveal new mechanisms adjusting cellular function to cope with hazardous environmental stress. Recent Advances: Key cell cycle regulators, including Cyclin D1/CDK4 and Cyclin B1/cyclin-dependent kinase 1 (CDK1) complexes, are actively involved in the regulation of mitochondrial functions via phosphorylation of their mitochondrial targets. Accumulating new evidence supports a concept that the Cyclin B1/CDK1 complex acts as a mediator in the cross talk between radiation-induced DNA damage and mitochondrial functions to coordinate cellular responses to low-level genotoxic stresses. Critical Issues: The LDIR-mediated mitochondrial activity via Cyclin B1/CDK1 regulation is an irreplaceable network that is able to harmonize vital cellular functions with adjusted mitochondrial metabolism to enhance cellular homeostasis. Future Directions: Further investigation of the coordinative mechanism that regulates mitochondrial activities in sublethal stress conditions, including LDIR, will reveal new insights of how cells cope with genotoxic injury and will be vital for future targeted therapeutic interventions that reduce environmental injury and cancer risk. Antioxid. Redox Signal. 20, 1463–1480. PMID:24180340

The role of transient receptor potential channels in joint diseases.

PubMed

Krupkova, O; Zvick, J; Wuertz-Kozak, K

2017-10-10

Transient receptor potential channels (TRP channels) are cation selective transmembrane receptors with diverse structures, activation mechanisms and physiological functions. TRP channels act as cellular sensors for a plethora of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimuli, pH and endogenous, as well as, exogenous ligands, thereby illustrating their versatility. As such, TRP channels regulate various functions in both excitable and non-excitable cells, mainly by mediating Ca2+ homeostasis. Dysregulation of TRP channels is implicated in many pathologies, including cardiovascular diseases, muscular dystrophies and hyperalgesia. However, the importance of TRP channel expression, physiological function and regulation in chondrocytes and intervertebral disc (IVD) cells is largely unexplored. Osteoarthritis (OA) and degenerative disc disease (DDD) are chronic age-related disorders that significantly affect the quality of life by causing pain, activity limitation and disability. Furthermore, currently available therapies cannot effectively slow-down or stop progression of these diseases. Both OA and DDD are characterised by reduced tissue cellularity, enhanced inflammatory responses and molecular, structural and mechanical alterations of the extracellular matrix, hence affecting load distribution and reducing joint flexibility. However, knowledge on how chondrocytes and IVD cells sense their microenvironment and respond to its changes is still limited. In this review, we introduced six families of mammalian TRP channels, their mechanisms of activation, as well as, activation-driven cellular consequences. We summarised the current knowledge on TRP channel expression and activity in chondrocytes and IVD cells, as well as, the significance of TRP channels as therapeutic targets for the treatment of OA and DDD.

Understanding Biological Regulation Through Synthetic Biology.

PubMed

Bashor, Caleb J; Collins, James J

2018-05-20

Engineering synthetic gene regulatory circuits proceeds through iterative cycles of design, building, and testing. Initial circuit designs must rely on often-incomplete models of regulation established by fields of reductive inquiry-biochemistry and molecular and systems biology. As differences in designed and experimentally observed circuit behavior are inevitably encountered, investigated, and resolved, each turn of the engineering cycle can force a resynthesis in understanding of natural network function. Here, we outline research that uses the process of gene circuit engineering to advance biological discovery. Synthetic gene circuit engineering research has not only refined our understanding of cellular regulation but furnished biologists with a toolkit that can be directed at natural systems to exact precision manipulation of network structure. As we discuss, using circuit engineering to predictively reorganize, rewire, and reconstruct cellular regulation serves as the ultimate means of testing and understanding how cellular phenotype emerges from systems-level network function.

The human NAD metabolome: Functions, metabolism and compartmentalization

PubMed Central

Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

2015-01-01

Abstract The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools. PMID:25837229

Severe Acneiform Eruption Induced by Cetuximab (Erbitux®)

PubMed Central

Lee, Jung Eun; Lee, Sang Ju; Lee, Hee Jung; Lee, Ju Hee

2008-01-01

Epidermal growth factor has an important role in the regulation of proliferation and differentiation in epidermal keratinocytes, as well as in the survival, angiogenesis and metastasis of cancer cells. Cetuximab is a chimeric monoclonal antibody selective for the epidermal growth factor receptor that induces a broad range of cellular responses that enhance tumor sensitivity to radiotherapy and chemotherapeutic agents. However, it can cause adverse events in the patient including acneiform eruption, asthenia, abdominal pain and nausea/vomiting. We report a case of severe acneiform eruption induced by cetuximab in a 56-year-old man with colorectal cancer and liver metastases. PMID:18972607

Optimizing Cellular Networks Enabled with Renewal Energy via Strategic Learning.

PubMed

Sohn, Insoo; Liu, Huaping; Ansari, Nirwan

2015-01-01

An important issue in the cellular industry is the rising energy cost and carbon footprint due to the rapid expansion of the cellular infrastructure. Greening cellular networks has thus attracted attention. Among the promising green cellular network techniques, the renewable energy-powered cellular network has drawn increasing attention as a critical element towards reducing carbon emissions due to massive energy consumption in the base stations deployed in cellular networks. Game theory is a branch of mathematics that is used to evaluate and optimize systems with multiple players with conflicting objectives and has been successfully used to solve various problems in cellular networks. In this paper, we model the green energy utilization and power consumption optimization problem of a green cellular network as a pilot power selection strategic game and propose a novel distributed algorithm based on a strategic learning method. The simulation results indicate that the proposed algorithm achieves correlated equilibrium of the pilot power selection game, resulting in optimum green energy utilization and power consumption reduction.

Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1

PubMed Central

Schwertassek, Ulla; Balmer, Yves; Gutscher, Marcus; Weingarten, Lars; Preuss, Marc; Engelhard, Johanna; Winkler, Monique; Dick, Tobias P

2007-01-01

The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor–ligand signaling interactions can be selectively regulated by an extracellular redox catalyst. PMID:17557078

Expression of a transmembrane phosphotyrosine phosphatase inhibits cellular response to platelet-derived growth factor and insulin-like growth factor-1.

PubMed

Mooney, R A; Freund, G G; Way, B A; Bordwell, K L

1992-11-25

Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.

NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

PubMed

Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

2018-01-20

The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis

PubMed Central

Knippschild, Uwe; Krüger, Marc; Richter, Julia; Xu, Pengfei; García-Reyes, Balbina; Peifer, Christian; Halekotte, Jakob; Bakulev, Vasiliy; Bischof, Joachim

2014-01-01

Members of the highly conserved and ubiquitously expressed pleiotropic CK1 family play major regulatory roles in many cellular processes including DNA-processing and repair, proliferation, cytoskeleton dynamics, vesicular trafficking, apoptosis, and cell differentiation. As a consequence of cellular stress conditions, interaction of CK1 with the mitotic spindle is manifold increased pointing to regulatory functions at the mitotic checkpoint. Furthermore, CK1 is able to alter the activity of key proteins in signal transduction and signal integration molecules. In line with this notion, CK1 is tightly connected to the regulation and degradation of β-catenin, p53, and MDM2. Considering the importance of CK1 for accurate cell division and regulation of tumor suppressor functions, it is not surprising that mutations and alterations in the expression and/or activity of CK1 isoforms are often detected in various tumor entities including cancer of the kidney, choriocarcinomas, breast carcinomas, oral cancer, adenocarcinomas of the pancreas, and ovarian cancer. Therefore, scientific effort has enormously increased (i) to understand the regulation of CK1 and its involvement in tumorigenesis- and tumor progression-related signal transduction pathways and (ii) to develop CK1-specific inhibitors for the use in personalized therapy concepts. In this review, we summarize the current knowledge regarding CK1 regulation, function, and interaction with cellular proteins playing central roles in cellular stress-responses and carcinogenesis. PMID:24904820

Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly.

PubMed

Pham, Tu M; Tran, Si C; Lim, Yun-Sook; Hwang, Soon B

2017-02-01

Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly. Copyright © 2017 American Society for Microbiology.

Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15*

PubMed Central

Oberg, Elizabeth A.; Nifoussi, Shanna K.; Gingras, Anne-Claude; Strack, Stefan

2012-01-01

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (AC) bound to a variable (B) regulatory subunit. PP2A is an enzyme family of dozens of heterotrimers with different subcellular locations and cellular substrates dictated by the B subunit. B′β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca2+/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B′β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of ∼40 Kelch-like proteins, many of which have been identified as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. Here, we report that KLHL15-Cul3 specifically targets B′β to promote turnover of the PP2A subunit by ubiquitylation and proteasomal degradation. Comparison of KLHL15 and B′β tissue expression profiles suggests that the E3 ligase adaptor contributes to selective expression of the PP2A/B′β holoenzyme in the brain. We mapped KLHL15 residues critical for homodimerization as well as interaction with Cul3 and B′β. Explaining PP2A subunit selectivity, the divergent N terminus of B′β was found necessary and sufficient for KLHL15-mediated degradation, with Tyr-52 having an obligatory role. Although KLHL15 can interact with the PP2A/B′β heterotrimer, it only degrades B′β, thus promoting exchange with other regulatory subunits. E3 ligase adaptor-mediated control of PP2A holoenzyme composition thereby adds another layer of regulation to cellular dephosphorylation events. PMID:23135275

Tipping the balance of RNA stability by 3' editing of the transcriptome.

PubMed

Chung, Christina Z; Seidl, Lauren E; Mann, Mitchell R; Heinemann, Ilka U

2017-11-01

The regulation of active microRNAs (miRNAs) and maturation of messenger RNAs (mRNAs) that are competent for translation is a crucial point in the control of all cellular processes, with established roles in development and differentiation. Terminal nucleotidyltransferases (TNTases) are potent regulators of RNA metabolism. TNTases promote the addition of single or multiple nucleotides to an RNA transcript that can rapidly alter transcript stability. The well-known polyadenylation promotes transcript stability while the newly discovered but ubiquitious 3'-end polyuridylation marks RNA for degradation. Monoadenylation and uridylation are essential control mechanisms balancing mRNA and miRNA homeostasis. This review discusses the multiple functions of non-canonical TNTases, focusing on their substrate range, biological functions, and evolution. TNTases directly control mRNA and miRNA levels, with diverse roles in transcriptome stabilization, maturation, silencing, or degradation. We will summarize the current state of knowledge on non-canonical nucleotidyltransferases and their function in regulating miRNA and mRNA metabolism. We will review the discovery of uridylation as an RNA degradation pathway and discuss the evolution of nucleotidyltransferases along with their use in RNA labeling and future applications as therapeutic targets. The biochemically and evolutionarily highly related adenylyl- and uridylyltransferases play antagonizing roles in the cell. In general, RNA adenylation promotes stability, while uridylation marks RNA for degradation. Uridylyltransferases evolved from adenylyltransferases in multiple independent evolutionary events by the insertion of a histidine residue into the active site, altering nucleotide, but not RNA specificity. Understanding the mechanisms regulating RNA stability in the cell and controlling the transcriptome is essential for efforts aiming to influence cellular fate. Selectively enhancing or reducing RNA stability allows for alterations in the transcriptome, proteome, and downstream cellular processes. Genetic, biochemical, and clinical data suggest TNTases are potent targets for chemotherapeutics and have been exploited for RNA labeling applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

A mitochondria-selective near-infrared-emitting fluorescent dye for cellular imaging studies.

PubMed

Choi, Peter; Noguchi, Katsuya; Ishiyama, Munetaka; Denny, William A; Jose, Jiney

2018-05-03

This communication details the synthesis, evaluation of photophysical properties, and cellular imaging studies of cyanine chromophore based fluorescent dye 1 as a selective imaging agent for mitochondria. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

NASA Astrophysics Data System (ADS)

Ivanova, Krassimira; Eiermann, Peter; Tsiockas, Wasiliki; Hemmersbach, Ruth; Gerzer, Rupert

2018-03-01

Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer.

PubMed

Zhao, Na; Cao, Jin; Xu, Longyong; Tang, Qianzi; Dobrolecki, Lacey E; Lv, Xiangdong; Talukdar, Manisha; Lu, Yang; Wang, Xiaoran; Hu, Dorothy Z; Shi, Qing; Xiang, Yu; Wang, Yunfei; Liu, Xia; Bu, Wen; Jiang, Yi; Li, Mingzhou; Gong, Yingyun; Sun, Zheng; Ying, Haoqiang; Yuan, Bo; Lin, Xia; Feng, Xin-Hua; Hartig, Sean M; Li, Feng; Shen, Haifa; Chen, Yiwen; Han, Leng; Zeng, Qingping; Patterson, John B; Kaipparettu, Benny Abraham; Putluri, Nagireddy; Sicheri, Frank; Rosen, Jeffrey M; Lewis, Michael T; Chen, Xi

2018-04-02

The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.

KDM5 Interacts with Foxo to Modulate Cellular Levels of Oxidative Stress

PubMed Central

Liu, Xingyin; Greer, Christina; Secombe, Julie

2014-01-01

Increased cellular levels of oxidative stress are implicated in a large number of human diseases. Here we describe the transcription co-factor KDM5 (also known as Lid) as a new critical regulator of cellular redox state. Moreover, this occurs through a novel KDM5 activity whereby it alters the ability of the transcription factor Foxo to bind to DNA. Our microarray analyses of kdm5 mutants revealed a striking enrichment for genes required to regulate cellular levels of oxidative stress. Consistent with this, loss of kdm5 results in increased sensitivity to treatment with oxidizers, elevated levels of oxidized proteins, and increased mutation load. KDM5 activates oxidative stress resistance genes by interacting with Foxo to facilitate its recruitment to KDM5-Foxo co-regulated genes. Significantly, this occurs independently of KDM5's well-characterized demethylase activity. Instead, KDM5 interacts with the lysine deacetylase HDAC4 to promote Foxo deacetylation, which affects Foxo DNA binding. PMID:25329053

β-Catenin recognizes a specific RNA motif in the cyclooxygenase-2 mRNA 3′-UTR and interacts with HuR in colon cancer cells

PubMed Central

Kim, Inae; Kwak, Hoyun; Lee, Hee Kyu; Hyun, Soonsil; Jeong, Sunjoo

2012-01-01

RNA-binding proteins regulate multiple steps of RNA metabolism through both dynamic and combined binding. In addition to its crucial roles in cell adhesion and Wnt-activated transcription in cancer cells, β-catenin regulates RNA alternative splicing and stability possibly by binding to target RNA in cells. An RNA aptamer was selected for specific binding to β-catenin to address RNA recognition by β-catenin more specifically. Here, we characterized the structural properties of the RNA aptamer as a model and identified a β-catenin RNA motif. Similar RNA motif was found in cellular RNA, Cyclooxygenase-2 (COX-2) mRNA 3′-untranslated region (3′-UTR). More significantly, the C-terminal domain of β-catenin interacted with HuR and the Armadillo repeat domain associated with RNA to form the RNA–β-catenin–HuR complex in vitro and in cells. Furthermore, the tertiary RNA–protein complex was predominantly found in the cytoplasm of colon cancer cells; thus, it might be related to COX-2 protein level and cancer progression. Taken together, the β-catenin RNA aptamer was valuable for deducing the cellular RNA aptamer and identifying novel and oncogenic RNA–protein networks in colon cancer cells. PMID:22544606

AIRE-induced apoptosis is associated with nuclear translocation of stress sensor protein GAPDH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liiv, Ingrid, E-mail: ingrid.liiv@ut.ee; Haljasorg, Uku; Kisand, Kai

2012-06-22

Highlights: Black-Right-Pointing-Pointer AIRE induces apoptosis in epithelial cells. Black-Right-Pointing-Pointer CARD domain of AIRE is sufficient for apoptosis induction. Black-Right-Pointing-Pointer AIRE induced apoptosis involves GAPDH translocation to the nuclei. Black-Right-Pointing-Pointer Deprenyl inhibits AIRE induced apoptosis. -- Abstract: AIRE (Autoimmune Regulator) has a central role in the transcriptional regulation of self-antigens in medullary thymic epithelial cells, which is necessary for negative selection of autoreactive T cells. Recent data have shown that AIRE can also induce apoptosis, which may be linked to cross-presentation of these self-antigens. Here we studied AIRE-induced apoptosis using AIRE over-expression in a thymic epithelial cell line as well asmore » doxycycline-inducible HEK293 cells. We show that the HSR/CARD domain in AIRE together with a nuclear localization signal is sufficient to induce apoptosis. In the nuclei of AIRE-positive cells, we also found an increased accumulation of a glycolytic enzyme, glyceraldehyde-3-phosphate (GAPDH) reflecting cellular stress and apoptosis. Additionally, AIRE-induced apoptosis was inhibited with an anti-apoptotic agent deprenyl that blocks GAPDH nitrosylation and nuclear translocation. We propose that the AIRE-induced apoptosis pathway is associated with GAPDH nuclear translocation and induction of NO-induced cellular stress in AIRE-expressing cells.« less

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

PubMed

Ailte, Ieva; Lingelem, Anne Berit Dyve; Kavaliauskiene, Simona; Bergan, Jonas; Kvalvaag, Audun Sverre; Myrann, Anne-Grethe; Skotland, Tore; Sandvig, Kirsten

2016-07-26

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

Plant Translation Factors and Virus Resistance

PubMed Central

Sanfaçon, Hélène

2015-01-01

Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable. PMID:26114476

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions*

PubMed Central

Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

2017-01-01

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database. Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se. Further, our data will have a significant impact on the ongoing debate about patient treatment modalities. PMID:28302921

β1-Integrin Deletion From the Lens Activates Cellular Stress Responses Leading to Apoptosis and Fibrosis

PubMed Central

Wang, Yichen; Terrell, Anne M.; Riggio, Brittany A.; Anand, Deepti; Lachke, Salil A.; Duncan, Melinda K.

2017-01-01

Purpose Previous research showed that the absence of β1-integrin from the mouse lens after embryonic day (E) 13.5 (β1MLR10) leads to the perinatal apoptosis of lens epithelial cells (LECs) resulting in severe microphthalmia. This study focuses on elucidating the molecular connections between β1-integrin deletion and this phenotype. Methods RNA sequencing was performed to identify differentially regulated genes (DRGs) in β1MLR10 lenses at E15.5. By using bioinformatics analysis and literature searching, Egr1 (early growth response 1) was selected for further study. The activation status of certain signaling pathways (focal adhesion kinase [FAK]/Erk, TGF-β, and Akt signaling) was studied via Western blot and immunohistochemistry. Mice lacking both β1-integrin and Egr1 genes from the lenses were created (β1MLR10/Egr1−/−) to study their relationship. Results RNA sequencing identified 120 DRGs that include candidates involved in the cellular stress response, fibrosis, and/or apoptosis. Egr1 was investigated in detail, as it mediates cellular stress responses in various cell types, and is recognized as an upstream regulator of numerous other β1MLR10 lens DRGs. In β1MLR10 mice, Egr1 levels are elevated shortly after β1-integrin loss from the lens. Further, pErk1/2 and pAkt are elevated in β1MLR10 LECs, thus providing the potential signaling mechanism that causes Egr1 upregulation in the mutant. Indeed, deletion of Egr1 from β1MLR10 lenses partially rescues the microphthalmia phenotype. Conclusions β1-integrin regulates the appropriate levels of Erk1/2 and Akt phosphorylation in LECs, whereas its deficiency results in the overexpression of Egr1, culminating in reduced cell survival. These findings provide insight into the molecular mechanism underlying the microphthalmia observed in β1MLR10 mice. PMID:28763805

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penrose, Harrison; Heller, Sandra; Cable, Chloe

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTORmore » and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.« less

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions.

PubMed

Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

2017-05-01

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database.Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments.In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se Further, our data will have a significant impact on the ongoing debate about patient treatment modalities. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Endoplasmic Reticulum-Plasma Membrane Contacts Regulate Cellular Excitability.

PubMed

Dickson, Eamonn J

2017-01-01

Cells that have intrinsic electrical excitability utilize changes in membrane potential to communicate with neighboring cells and initiate cellular cascades. Excitable cells like neurons and myocytes have evolved highly specialized subcellular architectures to translate these electrical signals into cellular events. One such structural specialization is sarco-/endoplasmic reticulum-plasma membrane contact sites. These membrane contact sites are positioned by specific membrane-membrane tethering proteins and contain an ever-expanding list of additional proteins that organize information transfer across the junctional space (~ 15-25 nm distance) to shape membrane identity and control cellular excitability. In this chapter we discuss how contacts between the sarco-/endoplasmic reticulum and plasma membrane are essential for regulated excitation-contraction coupling in striated muscle and control of lipid-dependent ion channels.

Endoplasmic reticulum turnover: ER-phagy and other flavors in selective and non-selective ER clearance.

PubMed

Fregno, Ilaria; Molinari, Maurizio

2018-01-01

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells. It is deputed to lipid and protein biosynthesis, calcium storage, and the detoxification of various exogenous and endogenous harmful compounds. ER activity and size must be adapted rapidly to environmental and developmental conditions or biosynthetic demand. This is achieved on induction of thoroughly studied transcriptional/translational programs defined as "unfolded protein responses" that increase the ER volume and the expression of ER-resident proteins regulating the numerous ER functions. Less understood are the lysosomal catabolic processes that maintain ER size at steady state, that prevent excessive ER expansion during ER stresses, or that ensure return to physiologic ER size during recovery from ER stresses. These catabolic processes may also be activated to remove ER subdomains where proteasome-resistant misfolded proteins or damaged lipids have been segregated. Insights into these catabolic mechanisms have only recently emerged with the identification of so-called ER-phagy receptors, which label specific ER subdomains for selective lysosomal delivery for clearance. Here, in eight chapters and one addendum, we comment on recent advances in ER turnover pathways induced by ER stress, nutrient deprivation, misfolded proteins, and live bacteria. We highlight the role of yeast (Atg39 and Atg40) and mammalian (FAM134B, SEC62, RTN3, and CCPG1) ER-phagy receptors and of autophagy genes in selective and non-selective catabolic processes that regulate cellular proteostasis by controlling ER size, turnover, and function.

Protein accounting in the cellular economy.

PubMed

Vázquez-Laslop, Nora; Mankin, Alexander S

2014-04-24

Knowing the copy number of cellular proteins is critical for understanding cell physiology. By being able to measure the absolute synthesis rates of the majority of cellular proteins, Li et al. gain insights into key aspects of translation regulation and fundamental principles of cellular strategies to adjust protein synthesis according to the functional needs. Copyright © 2014 Elsevier Inc. All rights reserved.

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion

PubMed Central

A.P., Sudheesh

2017-01-01

ABSTRACT Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3′-end processing and expression of pre-mRNAs encoding key anti-invasive factors (KISS1R, CDH1, NME1, CDH13, FEZ1, and WIF1) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP–PIPKIα-mediated 3′-end processing as a key anti-invasive mechanism in breast cancer. PMID:29203642

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion.

PubMed

A P, Sudheesh; Laishram, Rakesh S

2018-03-01

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors ( KISS1R , CDH1 , NME1 , CDH13 , FEZ1 , and WIF1 ) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP-PIPKIα-mediated 3'-end processing as a key anti-invasive mechanism in breast cancer. Copyright © 2018 A.P. and Laishram.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

PubMed

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-24

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an Apc Min/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior

PubMed Central

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-01

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion. PMID:28057861

AP1 Keeps Chromatin Poised for Action | Center for Cancer Research

Cancer.gov

The human genome harbors gene-encoding DNA, the blueprint for building proteins that regulate cellular function. Embedded across the genome, in non-coding regions, are DNA elements to which regulatory factors bind. The interaction of regulatory factors with DNA at these sites modifies gene expression to modulate cell activity. In cells, DNA exists in a complex with proteins called chromatin that compacts the DNA in the nucleus, strongly restricting access to DNA sequences. As a result, regulatory factors only interact with a small subset of their potential binding elements in a given cell to regulate genes. How factors recognize and select sites in chromatin across the genome is not well understood -- but several discoveries in CCR’s Laboratory of Receptor Biology and Gene Expression (LRBGE) have shed light on the mechanisms that direct factors to DNA.

Autophagic Regulation of Lipid Homeostasis in Cardiometabolic Syndrome.

PubMed

Yang, Mingjie; Zhang, Yingmei; Ren, Jun

2018-01-01

As an important protein quality control process, autophagy is essential for the degradation and removal of long-lived or injured cellular components and organelles. Autophagy is known to participate in a number of pathophysiological processes including cardiometabolic syndrome. Recent findings have shown compelling evidence for the intricate interplay between autophagy and lipid metabolism. Autophagy serves as a major regulator of lipid homeostasis while lipid can also influence autophagosome formation and autophagic signaling. Lipophagy is a unique form of selective autophagy and functions as a fundamental mechanism for clearance of lipid excess in atherosclerotic plaques. Ample of evidence has denoted a novel therapeutic potential for autophagy in deranged lipid metabolism and management of cardiometabolic diseases such as atherosclerosis and diabetic cardiomyopathy. Here we will review the interplays between cardiac autophagy and lipid metabolism in an effort to seek new therapeutic options for cardiometabolic diseases.

Breaking into the epithelial apical–junctional complex — news from pathogen hackers

PubMed Central

Vogelmann, Roger; Amieva, Manuel R; Falkow, Stanley; Nelson, W James

2012-01-01

The epithelial apical–junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical–junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical–junctional complex of the Ig superfamily — junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor — are important regulators of junction structure and function and represent critical targets of microbial virulence gene products. PMID:15037310

Breaking into the epithelial apical-junctional complex--news from pathogen hackers.

PubMed

Vogelmann, Roger; Amieva, Manuel R; Falkow, Stanley; Nelson, W James

2004-02-01

The epithelial apical-junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical-junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical-junctional complex of the Ig superfamily--junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor--are important regulators of junction structure and function and represent critical targets of microbial virulence gene products.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

PubMed

Bar-Peled, Liron; Kemper, Esther K; Suciu, Radu M; Vinogradova, Ekaterina V; Backus, Keriann M; Horning, Benjamin D; Paul, Thomas A; Ichu, Taka-Aki; Svensson, Robert U; Olucha, Jose; Chang, Max W; Kok, Bernard P; Zhu, Zhou; Ihle, Nathan T; Dix, Melissa M; Jiang, Ping; Hayward, Matthew M; Saez, Enrique; Shaw, Reuben J; Cravatt, Benjamin F

2017-10-19

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

PubMed Central

Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel

2017-01-01

ABSTRACT Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells. IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression. PMID:28331082

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Mitochondrial morphology transitions and functions: implications for retrograde signaling?

PubMed Central

Picard, Martin; Shirihai, Orian S.; Gentil, Benoit J.

2013-01-01

In response to cellular and environmental stresses, mitochondria undergo morphology transitions regulated by dynamic processes of membrane fusion and fission. These events of mitochondrial dynamics are central regulators of cellular activity, but the mechanisms linking mitochondrial shape to cell function remain unclear. One possibility evaluated in this review is that mitochondrial morphological transitions (from elongated to fragmented, and vice-versa) directly modify canonical aspects of the organelle's function, including susceptibility to mitochondrial permeability transition, respiratory properties of the electron transport chain, and reactive oxygen species production. Because outputs derived from mitochondrial metabolism are linked to defined cellular signaling pathways, fusion/fission morphology transitions could regulate mitochondrial function and retrograde signaling. This is hypothesized to provide a dynamic interface between the cell, its genome, and the fluctuating metabolic environment. PMID:23364527

Regulation of alternative mRNA splicing: old players and new perspectives.

PubMed

Dvinge, Heidi

2018-06-01

Nearly all human multi-exon genes are subject to alternative splicing in one or more cell types. The splicing machinery, therefore, has to select between multiple splice sites in a context-dependent manner, relying on sequence features in cis and trans-acting splicing regulators that either promote or repress splice site recognition and spliceosome assembly. However, the functional coupling between multiple gene regulatory layers signifies that splicing can also be modulated by transcriptional or epigenetic characteristics. Other, less obvious, aspects of alternative splicing have come to light in recent years, often involving core components of the spliceosome previously thought to perform a basal rather than a regulatory role in splicing. Together this paints a highly dynamic picture of splicing regulation, where the final splice site choice is governed by the entire transcriptional environment of a gene and its cellular context. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Methylation-regulated decommissioning of multimeric PP2A complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Cheng-Guo; Zheng, Aiping; Jiang, Li

2017-12-01

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivationmore » of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.« less

Mechanisms and regulation of DNA replication initiation in eukaryotes

PubMed Central

Parker, Matthew W.; Botchan, Michael R.; Berger, James M.

2017-01-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

Mechanisms and regulation of DNA replication initiation in eukaryotes.

PubMed

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Small RNA Profiling of Influenza A Virus-Infected Cells Identifies miR-449b as a Regulator of Histone Deacetylase 1 and Interferon Beta

PubMed Central

Buggele, William A.; Krause, Katherine E.; Horvath, Curt M.

2013-01-01

The mammalian antiviral response relies on the alteration of cellular gene expression, to induce the production of antiviral effectors and regulate their activities. Recent research has indicated that virus infections can induce the accumulation of cellular microRNA (miRNA) species that influence the stability of host mRNAs and their protein products. To determine the potential for miRNA regulation of cellular responses to influenza A virus infection, small RNA profiling was carried out using next generation sequencing. Comparison of miRNA expression profiles in uninfected human A549 cells to cells infected with influenza A virus strains A/Udorn/72 and A/WSN/33, revealed virus-induced changes in miRNA abundance. Gene expression analysis identified mRNA targets for a cohort of highly inducible miRNAs linked to diverse cellular functions. Experiments demonstrate that the histone deacetylase, HDAC1, can be regulated by influenza-inducible miR-449b, resulting in altered mRNA and protein levels. Expression of miR-449b enhances virus and poly(I:C) activation of the IFNβ promoter, a process known to be negatively regulated by HDAC1. These findings demonstrate miRNA induction by influenza A virus infection and elucidate an example of miRNA control of antiviral gene expression in human cells, defining a role for miR-449b in regulation of HDAC1 and antiviral cytokine signaling. PMID:24086750

Use of Mature miRNA Strand Selection in miRNAs Families in Cervical Cancer Development

PubMed Central

Granados-López, Angelica Judith; Ruiz-Carrillo, José Luis; Servín-González, Luis Steven; Martínez-Rodríguez, José Luis; Reyes-Estrada, Claudia Araceli; Gutiérrez-Hernández, Rosalinda; López, Jesús Adrián

2017-01-01

Aberrant miRNA expression is well recognized as a cancer hallmark, nevertheless miRNA function and expression does not always correlate in patients tissues and cell lines studies. In addition to this issue, miRNA strand usage conduces to increased cell signaling pathways modulation diversifying cellular processes regulation. In cervical cancer, 20 miRNA families are involved in carcinogenesis induction and development to this moment. These families have 5p and 3p strands with different nucleotide (nt) chain sizes. In general, mature 5p strands are larger: two miRNAs of 24 nt, 24 miRNAs of 23 nt, 35 miRNAs of 22 nt and three miRNAs of 21 nt. On the other hand, the 3p strands lengths observed are: seven miRNAs of 23 nt, 50 miRNAs of 22 nt, six miRNAs of 21 nt and four miRNAs of 20 nt. Based on the analysis of the 20 miRNA families associated with cervical cancer, 67 3p strands and 65 5p strands are selected suggesting selectivity and specificity mechanisms regulating cell processes like proliferation, apoptosis, migration, invasion, metabolism and Warburg effect. The insight reviewed here could be used in the miRNA based therapy, diagnosis and prognosis approaches. PMID:28216603

Use of Mature miRNA Strand Selection in miRNAs Families in Cervical Cancer Development.

PubMed

Granados-López, Angelica Judith; Ruiz-Carrillo, José Luis; Servín-González, Luis Steven; Martínez-Rodríguez, José Luis; Reyes-Estrada, Claudia Araceli; Gutiérrez-Hernández, Rosalinda; López, Jesús Adrián

2017-02-14

Aberrant miRNA expression is well recognized as a cancer hallmark, nevertheless miRNA function and expression does not always correlate in patients tissues and cell lines studies. In addition to this issue, miRNA strand usage conduces to increased cell signaling pathways modulation diversifying cellular processes regulation. In cervical cancer, 20 miRNA families are involved in carcinogenesis induction and development to this moment. These families have 5p and 3p strands with different nucleotide (nt) chain sizes. In general, mature 5p strands are larger: two miRNAs of 24 nt, 24 miRNAs of 23 nt, 35 miRNAs of 22 nt and three miRNAs of 21 nt. On the other hand, the 3p strands lengths observed are: seven miRNAs of 23 nt, 50 miRNAs of 22 nt, six miRNAs of 21 nt and four miRNAs of 20 nt. Based on the analysis of the 20 miRNA families associated with cervical cancer, 67 3p strands and 65 5p strands are selected suggesting selectivity and specificity mechanisms regulating cell processes like proliferation, apoptosis, migration, invasion, metabolism and Warburg effect. The insight reviewed here could be used in the miRNA based therapy, diagnosis and prognosis approaches.

Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration.

PubMed

Louie, Ke'ale W; Saera-Vila, Alfonso; Kish, Phillip E; Colacino, Justin A; Kahana, Alon

2017-11-09

Tissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature "post-mitotic" myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration. Following subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry. Reprogramming of a "post-mitotic" myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.

The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes.

PubMed

Wang, Hong-Mei; Xu, Yun-Fei; Ning, Shang-Lei; Yang, Du-Xiao; Li, Yi; Du, Yu-Jie; Yang, Fan; Zhang, Ya; Liang, Nan; Yao, Wei; Zhang, Ling-Li; Gu, Li-Chuan; Gao, Cheng-Jiang; Pang, Qi; Chen, Yu-Xin; Xiao, Kun-Hong; Ma, Rong; Yu, Xiao; Sun, Jin-Peng

2014-09-01

The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY(1112), the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12.

Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-15-1-0419 TITLE: Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease...COVERED 1 Sep 2016 - 31 Aug 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal...inappropriate cell growth, fluid secretion, and dysregulation of cellular energy metabolism. The enzyme AMPK regulates a number of cellular pathways, including

Role and regulation of autophagy in heat stress responses of tomato plants

PubMed Central

Zhou, Jie; Wang, Jian; Yu, Jing-Quan; Chen, Zhixiang

2014-01-01

As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG) genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing (VIGS) of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7, or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates. PMID:24817875

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

PubMed

Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-04-29

Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Ribosomal L1 domain and lysine-rich region are essential for CSIG/ RSL1D1 to regulate proliferation and senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Liwei; Zhao, Wenting; Zheng, Quanhui

2016-01-15

The expression change of cellular senescence-associated genes is underlying the genetic foundation of cellular senescence. Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) as a novel senescence-associated gene. CSIG is implicated in various process including cell cycle regulation, apoptosis, and tumor metastasis. We previously showed that CSIG plays an important role in regulating cell proliferation and cellular senescence progression through inhibiting PTEN, however, which domain or region of CSIG contributes to this function? To clarify this question, we investigated the functional importance of ribosomal L1 domain and lysine (Lys) -rich region of CSIG. Themore » data showed that expression of CSIG potently reduced PTEN expression, increased cell proliferation rates, and reduced the senescent phenotype (lower SA-β-gal activity). By contrast, neither the expression of CSIG N- terminal (NT) fragment containing the ribosomal L1 domain nor C-terminal (CT) fragment containing Lys-rich region could significantly altered the levels of PTEN; instead of promoting cell proliferation and delaying cellular senescence, expression of CSIG-NT or CSIG-CT inhibited cell proliferation and accelerated cell senescence (increased SA-β-gal activity) compared to either CSIG over-expressing or control (empty vector transfected) cells. The further immunofluorescence analysis showed that CSIG-CT and CSIG-NT truncated proteins exhibited different subcellular distribution with that of wild-type CSIG. Conclusively, both ribosomal L1 domain and Lys-rich region of CSIG are critical for CSIG to act as a regulator of cell proliferation and cellular senescence. - Highlights: • The ribosomal L1 domain and lysine-rich region of CSIG were expressed. • They are critical for CSIG to regulate proliferation and senescence. • CSIG and its domains exhibit different subcellular distribution.« less

A Unique Fungal Two-Component System Regulates Stress Responses, Drug Sensitivity, Sexual Development, and Virulence of Cryptococcus neoformans

PubMed Central

Bahn, Yong-Sun; Kojima, Kaihei; Cox, Gary M.

2006-01-01

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal “two-component” system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component–like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen. PMID:16672377

Microfabricated Nanotopological Surfaces for Study of Adhesion-dependent Cell mechanosensitivity**

PubMed Central

Chen, Weiqiang; Sun, Yubing

2014-01-01

Cells display high sensitivity and exhibit diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. Here, we reported a simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces using photolithography and reactive ion etching (RIE). Using RIE-generated nanorough glass surfaces, we demonstrated that local nanoroughness could provide a potent biophysical signal to regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation and migration. We further showed that cellular responses to nanotopography might be regulated by cell adhesion signaling and actin cytoskeleton remodeling. To further investigate the role of cytoskeleton contractility in nanoroughness sensing, we applied the RIE method to generate nanoroughness on the tops of an array of elastomeric poly-dimethylsiloxane (PDMS) microposts. We utilized the PDMS microposts as force sensors and demonstrated that nanoroughness could indeed regulate the cytoskeleton contractility of NIH/3T3 fibroblasts. Our results suggested that a feedback regulation and mechano-chemical integration mechanism involving adhesion signaling, actin cytoskeleton, and intracellular mechanosensory components might play an important role in regulating mechanosensitive behaviors of NIH/3T3 fibroblasts. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structures in vivo and suitable local cellular microenvironments for functional tissue engineering. PMID:22887768

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

PubMed

Jin, Hye Jin; Kwon, Ji Hye; Kim, Miyeon; Bae, Yun Kyung; Choi, Soo Jin; Oh, Wonil; Yang, Yoon Sun; Jeon, Hong Bae

2016-04-01

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs. One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy. ©AlphaMed Press.

The regulatory software of cellular metabolism.

PubMed

Segrè, Daniel

2004-06-01

Understanding the regulation of metabolic pathways in the cell is like unraveling the 'software' that is running on the 'hardware' of the metabolic network. Transcriptional regulation of enzymes is an important component of this software. A recent systematic analysis of metabolic gene-expression data in Saccharomyces cerevisiae reveals a complex modular organization of co-expressed genes, which could increase our ability to understand and engineer cellular metabolic functions.

Beyond laser microdissection technology: follow the yellow brick road for cancer research

PubMed Central

Legres, Luc G; Janin, Anne; Masselon, Christophe; Bertheau, Philippe

2014-01-01

Normal biological tissues harbour different populations of cells with intricate spacial distribution patterns resulting in heterogeneity of their overall cellular composition. Laser microdissection involving direct viewing and expertise by a pathologist, enables access to defined cell populations or specific region on any type of tissue sample, thus selecting near-pure populations of targeted cells. It opens the way for molecular methods directed towards well-defined populations, and provides also a powerful tool in studies focused on a limited number of cells. Laser microdissection has wide applications in oncology (diagnosis and research), cellular and molecular biology, biochemistry and forensics for tissue selection, but other areas have been gradually opened up to these new methodological approaches, such as cell cultures and cytogenetics. In clinical oncology trials, molecular profiling of microdissected samples can yield global “omics” information which, together, with the morphological analysis of cells, can provide the basis for diagnosis, prognosis and patient-tailored treatments. This remarkable technology has brought new insights in the understanding of DNA, RNA, and the biological functions and regulation of proteins to identify molecular disease signatures. We review herein the different applications of laser microdissection in a variety of fields, and we particularly focus attention on the pre-analytical steps that are crucial to successfully perform molecular-level investigations. PMID:24482735

A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae: Siw14 PROTEIN SELECTIVELY CLEAVES THE β-PHOSPHATE FROM 5-DIPHOSPHOINOSITOL PENTAKISPHOSPHATE (5PP-IP5).

PubMed

Steidle, Elizabeth A; Chong, Lucy S; Wu, Mingxuan; Crooke, Elliott; Fiedler, Dorothea; Resnick, Adam C; Rolfes, Ronda J

2016-03-25

Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, theSaccharomyces cerevisiaehomolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5or IP7)in vitro. In vivo,siw14Δ yeast mutants possess increased IP7levels, whereas heterologousSIW14overexpression eliminates IP7from cells. IP7levels increased proportionately whensiw14Δ was combined withddp1Δ orvip1Δ, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP7isoform 5PP-IP5to IP6. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of ROCK Activity in Cancer

PubMed Central

Morgan-Fisher, Marie; Wewer, Ulla M.

2013-01-01

Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)–loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer. PMID:23204112

High motivation for exercise is associated with altered chromatin regulators of monoamine receptor gene expression in the striatum of selectively bred mice.

PubMed

Saul, M C; Majdak, P; Perez, S; Reilly, M; Garland, T; Rhodes, J S

2017-03-01

Although exercise is critical for health, many lack the motivation to exercise, and it is unclear how motivation might be increased. To uncover the molecular underpinnings of increased motivation for exercise, we analyzed the transcriptome of the striatum in four mouse lines selectively bred for high voluntary wheel running and four non-selected control lines. The striatum was dissected and RNA was extracted and sequenced from four individuals of each line. We found multiple genes and gene systems with strong relationships to both selection and running history over the previous 6 days. Among these genes were Htr1b, a serotonin receptor subunit and Slc38a2, a marker for both glutamatergic and γ-aminobutyric acid (GABA)-ergic signaling. System analysis of the raw results found enrichment of transcriptional regulation and kinase genes. Further, we identified a splice variant affecting the Wnt-related Golgi signaling gene Tmed5. Using coexpression network analysis, we found a cluster of interrelated coexpression modules with relationships to running behavior. From these modules, we built a network correlated with running that predicts a mechanistic relationship between transcriptional regulation by nucleosome structure and Htr1b expression. The Library of Integrated Network-Based Cellular Signatures identified the protein kinase C δ inhibitor, rottlerin, the tyrosine kinase inhibitor, Linifanib and the delta-opioid receptor antagonist 7-benzylidenenaltrexone as potential compounds for increasing the motivation to run. Taken together, our findings support a neurobiological framework of exercise motivation where chromatin state leads to differences in dopamine signaling through modulation of both the primary neurotransmitters glutamate and GABA, and by neuromodulators such as serotonin. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors*

PubMed Central

Smith, Jeffrey S.; Rajagopal, Sudarshan

2016-01-01

The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. PMID:26984408

Systemic Activin signaling independently regulates sugar homeostasis, cellular metabolism, and pH balance in Drosophila melanogaster

PubMed Central

Ghosh, Arpan C.; O’Connor, Michael B.

2014-01-01

The ability to maintain cellular and physiological metabolic homeostasis is key for the survival of multicellular organisms in changing environmental conditions. However, our understanding of extracellular signaling pathways that modulate metabolic processes remains limited. In this study we show that the Activin-like ligand Dawdle (Daw) is a major regulator of systemic metabolic homeostasis and cellular metabolism in Drosophila. We find that loss of canonical Smad signaling downstream of Daw leads to defects in sugar and systemic pH homeostasis. Although Daw regulates sugar homeostasis by positively influencing insulin release, we find that the effect of Daw on pH balance is independent of its role in insulin signaling and is caused by accumulation of organic acids that are primarily tricarboxylic acid (TCA) cycle intermediates. RNA sequencing reveals that a number of TCA cycle enzymes and nuclear-encoded mitochondrial genes including genes involved in oxidative phosphorylation and β-oxidation are up-regulated in the daw mutants, indicating either a direct or indirect role of Daw in regulating these genes. These findings establish Activin signaling as a major metabolic regulator and uncover a functional link between TGF-β signaling, insulin signaling, and metabolism in Drosophila. PMID:24706779

Cellular and physiological mechanisms underlying blood flow regulation in the retina choroid in health disease

PubMed Central

Kur, Joanna; Newman, Eric A.; Chan-Ling, Tailoi

2012-01-01

We review the cellular and physiological mechanisms responsible for the regulation of blood flow in the retina and choroid in health and disease. Due to the intrinsic light sensitivity of the retina and the direct visual accessibility of fundus blood vessels, the eye offers unique opportunities for the non-invasive investigation of mechanisms of blood flow regulation. The ability of the retinal vasculature to regulate its blood flow is contrasted with the far more restricted ability of the choroidal circulation to regulate its blood flow by virtue of the absence of glial cells, the markedly reduced pericyte ensheathment of the choroidal vasculature, and the lack of intermediate filaments in choroidal pericytes. We review the cellular and molecular components of the neurovascular unit in the retina and choroid, techniques for monitoring retinal and choroidal blood flow, responses of the retinal and choroidal circulation to light stimulation, the role of capillaries, astrocytes and pericytes in regulating blood flow, putative signaling mechanisms mediating neurovascular coupling in the retina, and changes that occur in the retinal and choroidal circulation during diabetic retinopathy, age-related macular degeneration, glaucoma, and Alzheimer's disease. We close by discussing issues that remain to be explored. PMID:22580107

Yeast aquaporin regulation by 4-hydroxynonenal is implicated in oxidative stress response.

PubMed

Rodrigues, Claudia; Tartaro Bujak, Ivana; Mihaljević, Branka; Soveral, Graça; Cipak Gasparovic, Ana

2017-05-01

Reactive oxygen species, especially hydrogen peroxide (H 2 O 2 ), contribute to functional molecular impairment and cellular damage, but also are necessary in normal cellular metabolism, and in low doses play stimulatory role in cell proliferation and stress resistance. In parallel, reactive aldehydes such as 4-hydroxynonenal (HNE), are lipid peroxidation breakdown products which also contribute to regulation of numerous cellular processes. Recently, channeling of H 2 O 2 by some mammalian aquaporin isoforms has been reported and suggested to contribute to aquaporin involvement in cancer malignancies, although the mechanism by which these membrane water channels are implicated in oxidative stress is not clear. In this study, two yeast models with increased levels of membrane polyunsaturated fatty acids (PUFAs) and aquaporin AQY1 overexpression, respectively, were used to evaluate their interplay in cell's oxidative status. In particular, the aim of the study was to investigate if HNE accumulation could affect aquaporin function with an outcome in oxidative stress response. The data showed that induction of aquaporin expression by PUFAs results in increased water permeability in yeast membranes and that AQY1 activity is impaired by HNE. Moreover, AQY1 expression increases cellular sensitivity to oxidative stress by facilitating H 2 O 2 influx. On the other hand, AQY1 expression has no influence on the cellular antioxidant GSH levels and catalase activity. These results strongly suggest that aquaporins are important players in oxidative stress response and could contribute to regulation of cellular processes by regulation of H 2 O 2 influx. © 2017 IUBMB Life, 69(5):355-362, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Mapping of oxidative stress response elements of the caveolin-1 promoter.

PubMed

Bartholomew, Janine N; Galbiati, Ferruccio

2010-01-01

According to the "free radical theory" of aging, normal aging occurs as the result of tissue damages inflicted by reactive oxygen species (ROS). ROS are known to induce cellular senescence, and senescent cells are believed to contribute to organismal aging. The molecular mechanisms that mediate the cellular response to oxidants remain to be fully identified. We have shown that oxidative stress induces cellular senescence through activation of the caveolin-1 promoter and upregulation of caveolin-1 protein expression. Here, we describe how reactive oxygen species activate the caveolin-1 promoter and how the signaling may be assayed. These approaches provide insight into the functional role of caveolin-1 and potentially allow the identification of novel ROS-regulated genes that are part of the signaling machinery regulating cellular senescence/aging.

DUSP11 – An RNA phosphatase that regulates host and viral non-coding RNAs in mammalian cells

PubMed Central

Burke, James M.; Sullivan, Christopher S.

2017-01-01

ABSTRACT Dual-specificity phosphatase 11 (DUSP11) is a conserved protein tyrosine phosphatase (PTP) in metazoans. The cellular substrates and physiologic activities of DUSP11 remain largely unknown. In nematodes, DUSP11 is required for normal development and RNA interference against endogenous RNAs (endo-RNAi) via molecular mechanisms that are not well understood. However, mammals lack analogous endo-RNAi pathways and consequently, a role for DUSP11 in mammalian RNA silencing was unanticipated. Recent work from our laboratory demonstrated that DUSP11 activity alters the silencing potential of noncanonical viral miRNAs in mammalian cells. Our studies further uncovered direct cellular substrates of DUSP11 and suggest that DUSP11 is part of regulatory pathway that controls the abundance of select triphosphorylated noncoding RNAs. Here, we highlight recent findings and present new data that advance understanding of mammalian DUSP11 during gene silencing and discuss the emerging biological activities of DUSP11 in mammalian cells. PMID:28296624

Disruption of Hox9,10,11 function results in cellular level lineage infidelity in the kidney.

PubMed

Drake, Keri A; Adam, Mike; Mahoney, Robert; Potter, S Steven

2018-04-20

Hox genes are important regulators of development. The 39 mammalian Hox genes have considerable functional overlap, greatly confounding their study. In this report, we generated mice with multiple combinations of paralogous and flanking Abd-B Hox gene mutations to investigate functional redundancies in kidney development. The resulting mice developed a number of kidney abnormalities, including hypoplasia, agenesis, and severe cysts, with distinct Hox functions observed in early metanephric kidney formation and nephron progenitor maintenance. Most surprising, however, was that extensive removal of Hox shared function in these kidneys resulted in cellular level lineage infidelity. Strikingly, mutant nephron tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment. These results indicate that Hox genes are required for proper lineage selection/maintenance and full repression of genes involved in cell fate restriction in the developing kidney.

Interaction of carbon nanohorns with plants: Uptake and biological effects

DOE PAGES

Lahiani, Mohamed H.; Chen, Jihua; Irin, Fahmida; ...

2014-10-07

Single-Walled Carbon Nanohorns (SWCNHs) are a unique carbon-based nanomaterial with promising application in different fields including, medicine, genetic engineering and horticulture. Here, we investigated the biological response of six crop species (barley, corn, rice, soybean, switchgrass, tomato) and tobacco cell culture to the exposure of SWCNHs. We found that SWCNHs can activate seed germination of selected crops and enhance growth of different organs of corn, tomato, rice and soybean. At cellular level, growth of tobacco cells was increased in response to exposure of SWCNHs (78% increase compared to control). Uptake of SWCNHs by exposed crops and tobacco cells was confirmedmore » by transmission electron microscopy (TEM) and quantified by microwave induced heating (MIH) technique. At genetic level, SWCNHs were able to affect expression of a number of tomato genes that are involved in stress responses, cellular responses and metabolic processes. Our conclusion is that SWCNHs can be used as plant growth regulators and have the potential for plant-related applications.« less

Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila

PubMed Central

Yuan, Quan; Song, Yuanquan; Yang, Chung-Hui; Jan, Lily Yeh; Jan, Yuh Nung

2014-01-01

Intraspecific male-male aggression, important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, we established a behavioral paradigm in Drosophila melanogaster and investigated the relationship between sexual experience and aggression. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel, ppk29, and was mediated by male specific GABAergic neurons acting upon GABA-a receptor RDL in target cells. Silencing or activation of this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. We propose that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression. PMID:24241395

Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli

PubMed Central

Swings, Toon; Van den Bergh, Bram; Wuyts, Sander; Oeyen, Eline; Voordeckers, Karin; Verstrepen, Kevin J; Fauvart, Maarten; Verstraeten, Natalie; Michiels, Jan

2017-01-01

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.22939.001 PMID:28460660

Allosteric conformational barcodes direct signaling in the cell.

PubMed

Nussinov, Ruth; Ma, Buyong; Tsai, Chung-Jung; Csermely, Peter

2013-09-03

The cellular network is highly interconnected. Pathways merge and diverge. They proceed through shared proteins and may change directions. How are cellular pathways controlled and their directions decided, coded, and read? These questions become particularly acute when we consider that a small number of pathways, such as signaling pathways that regulate cell fates, cell proliferation, and cell death in development, are extensively exploited. This review focuses on these signaling questions from the structural standpoint and discusses the literature in this light. All co-occurring allosteric events (including posttranslational modifications, pathogen binding, and gain-of-function mutations) collectively tag the protein functional site with a unique barcode. The barcode shape is read by an interacting molecule, which transmits the signal. A conformational barcode provides an intracellular address label, which selectively favors binding to one partner and quenches binding to others, and, in this way, determines the pathway direction, and, eventually, the cell's response and fate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nuclear matrix - structure, function and pathogenesis.

PubMed

Wasąg, Piotr; Lenartowski, Robert

2016-12-20

The nuclear matrix (NM), or nuclear skeleton, is the non-chromatin, ribonucleoproteinaceous framework that is resistant to high ionic strength buffers, nonionic detergents, and nucleolytic enzymes. The NM fulfills a structural role in eukaryotic cells and is responsible for maintaining the shape of the nucleus and the spatial organization of chromatin. Moreover, the NM participates in several cellular processes, such as DNA replication/repair, gene expression, RNA transport, cell signaling and differentiation, cell cycle regulation, apoptosis and carcinogenesis. Short nucleotide sequences called scaffold/matrix attachment regions (S/MAR) anchor the chromatin loops to the NM proteins (NMP). The NMP composition is dynamic and depends on the cell type and differentiation stage or metabolic activity. Alterations in the NMP composition affect anchoring of the S/MARs and thus alter gene expression. This review aims to systematize information about the skeletal structure of the nucleus, with particular emphasis on the organization of the NM and its role in selected cellular processes. We also discuss several diseases that are caused by aberrant NM structure or dysfunction of individual NM elements.

The effects of selected drugs and dietary compounds on proliferation and apoptosis in colorectal carcinoma.

PubMed

Kiedrowski, Miroslaw; Mroz, Andrzej

2014-01-01

Like many malignancies, the development of colorectal carcinoma (CRC) can be considered as an imbalance between the compromised process of programmed cell death (apoptosis) and excessive, uncontrolled proliferation. Several mutations and epigenetic alterations are acquired during colorectal carcinogenesis. These are responsible for the cell cycle regulation, cellular sensitivity to pro- and antiapoptotic factors, cell proliferation, angiogenesis, invasiveness, as well as metastatic potential. The molecular alterations, along with their morphological expressions, have been recognised in detail, and most of the CRC cases can be attributed to either adenoma-carcinoma or serrated neoplasia pathways: in the first, the antiapoptotic features prevail; while in the second, the proliferative activity is of the utmost importance. The aim of the work is to discuss the influence of selected drugs and dietary compounds on the proliferation and apoptosis in CRC.

Selective disruption of the AKAP signaling complexes.

PubMed

Kennedy, Eileen J; Scott, John D

2015-01-01

Synthesis of the second messenger cAMP activates a variety of signaling pathways critical for all facets of intracellular regulation. Protein kinase A (PKA) is the major cAMP-responsive effector. Where and when this enzyme is activated has profound implications on the cellular role of PKA. A-Kinase Anchoring Proteins (AKAPs) play a critical role in this process by orchestrating spatial and temporal aspects of PKA action. A popular means of evaluating the impact of these anchored signaling events is to biochemically interfere with the PKA-AKAP interface. Hence, peptide disruptors of PKA anchoring are valuable tools in the investigation of local PKA action. This article outlines the development of PKA isoform-selective disruptor peptides, documents the optimization of cell-soluble peptide derivatives, and introduces alternative cell-based approaches that interrogate other aspects of the PKA-AKAP interface.

Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

PubMed Central

Karp, Cristina M.; Tan, Ting Ting; Mathew, Robin; Nelson, Deidre; Mukherjee, Chandreyee; Degenhardt, Kurt; Karantza-Wadsworth, Vassiliki; White, Eileen

2009-01-01

Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial (iBMK) cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity and contact inhibition, while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. PMID:18519669

A specific immune transcriptomic profile discriminates chronic kidney disease patients in predialysis from hemodialyzed patients

PubMed Central

2013-01-01

Background Chronic kidney disease (CKD) patients present a complex interaction between the innate and adaptive immune systems, in which immune activation (hypercytokinemia and acute-phase response) and immune suppression (impairment of response to infections and poor development of adaptive immunity) coexist. In this setting, circulating uremic toxins and microinflammation play a critical role. This condition, already present in the last stages of renal damage, seems to be enhanced by the contact of blood with bioincompatible extracorporeal hemodialysis (HD) devices. However, although largely described, the cellular machinery associated to the CKD- and HD-related immune-dysfunction is still poorly defined. Understanding the mechanisms behind this important complication may generate a perspective for improving patients outcome. Methods To better recognize the biological bases of the CKD-related immune dysfunction and to identify differences between CKD patients in conservative (CKD) from those in HD treatment, we used an high-throughput strategy (microarray) combined with classical bio-molecular approaches. Results Immune transcriptomic screening of peripheral blood mononuclear cells (1030 gene probe sets selected by Gene-Ontology) showed that 275 gene probe sets (corresponding to 213 genes) discriminated 9 CKD patients stage III-IV (mean ± SD of eGFR: 32.27±14.7 ml/min) from 17 HD patients (p < 0.0001, FDR = 5%). Seventy-one genes were up- and 142 down-regulated in HD patients. Functional analysis revealed, then, close biological links among the selected genes with a pivotal role of PTX3, IL-15 (up-regulated in HD) and HLA-G (down-regulated in HD). ELISA, performed on an independent testing-group [11 CKD stage III-IV (mean ± SD of eGFR: 30.26±14.89 ml/min) and 13 HD] confirmed that HLA-G, a protein with inhibition effects on several immunological cell lines including natural killers (NK), was down-expressed in HD (p = 0.04). Additionally, in the testing-group, protein levels of CX3CR1, an highly selective chemokine receptor and surface marker for cytotoxic effector lymphocytes, resulted higher expressed in HD compared to CKD (p < 0.01). Conclusion Taken together our results show, for the first time, that HD patients present a different immune-pattern compared to the un-dialyzed CKD patients. Among the selected genes, some of them encode for important biological elements involved in proliferation/activation of cytotoxic effector lymphocytes and in the immune-inflammatory cellular machinery. Additionally, this study reveals new potential diagnostic bio-markers and therapeutic targets. PMID:23663527

Development of Pyrazolone and Isoxazol-5-one Cambinol Analogues as Sirtuin Inhibitors

PubMed Central

2015-01-01

Sirtuins are a family of NAD+-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition. PMID:24697269

Regulation of ROS Production and Vascular Function by Carbon Monoxide

PubMed Central

Choi, Yoon Kyung; Por, Elaine D.; Kwon, Young-Guen; Kim, Young-Myeong

2012-01-01

Carbon monoxide (CO) is a gaseous molecule produced from heme by heme oxygenase (HO). CO interacts with reduced iron of heme-containing proteins, leading to its involvement in various cellular events via its production of mitochondrial reactive oxygen species (ROS). CO-mediated ROS production initiates intracellular signal events, which regulate the expression of adaptive genes implicated in oxidative stress and functions as signaling molecule for promoting vascular functions, including angiogenesis and mitochondrial biogenesis. Therefore, CO generated either by exogenous delivery or by HO activity can be fundamentally involved in regulating mitochondria-mediated redox cascades for adaptive gene expression and improving blood circulation (i.e., O2 delivery) via neovascularization, leading to the regulation of mitochondrial energy metabolism. This paper will highlight the biological effects of CO on ROS generation and cellular redox changes involved in mitochondrial metabolism and angiogenesis. Moreover, cellular mechanisms by which CO is exploited for disease prevention and therapeutic applications will also be discussed. PMID:22928087

Insights into the fluoride-resistant regulation mechanism of Acidithiobacillus ferrooxidans ATCC 23270 based on whole genome microarrays.

PubMed

Ma, Liyuan; Li, Qian; Shen, Li; Feng, Xue; Xiao, Yunhua; Tao, Jiemeng; Liang, Yili; Yin, Huaqun; Liu, Xueduan

2016-10-01

Acidophilic microorganisms involved in uranium bioleaching are usually suppressed by dissolved fluoride ions, eventually leading to reduced leaching efficiency. However, little is known about the regulation mechanisms of microbial resistance to fluoride. In this study, the resistance of Acidithiobacillus ferrooxidans ATCC 23270 to fluoride was investigated by detecting bacterial growth fluctuations and ferrous or sulfur oxidation. To explore the regulation mechanism, a whole genome microarray was used to profile the genome-wide expression. The fluoride tolerance of A. ferrooxidans cultured in the presence of FeSO4 was better than that cultured with the S(0) substrate. The differentially expressed gene categories closely related to fluoride tolerance included those involved in energy metabolism, cellular processes, protein synthesis, transport, the cell envelope, and binding proteins. This study highlights that the cellular ferrous oxidation ability was enhanced at the lower fluoride concentrations. An overview of the cellular regulation mechanisms of extremophiles to fluoride resistance is discussed.

NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward.

PubMed

Logan, Ryan W; Parekh, Puja K; Kaplan, Gabrielle N; Becker-Krail, Darius D; Williams, Wilbur P; Yamaguchi, Shintaro; Yoshino, Jun; Shelton, Micah A; Zhu, Xiyu; Zhang, Hui; Waplinger, Spencer; Fitzgerald, Ethan; Oliver-Smith, Jeffrey; Sundarvelu, Poornima; Enwright, John F; Huang, Yanhua H; McClung, Colleen A

2018-05-04

The diurnal regulation of dopamine is important for normal physiology and diseases such as addiction. Here we find a novel role for the CLOCK protein to antagonize CREB-mediated transcriptional activity at the tyrosine hydroxylase (TH) promoter, which is mediated by the interaction with the metabolic sensing protein, Sirtuin 1 (SIRT1). Additionally, we demonstrate that the transcriptional activity of TH is modulated by the cellular redox state, and daily rhythms of redox balance in the ventral tegmental area (VTA), along with TH transcription, are highly disrupted following chronic cocaine administration. Furthermore, CLOCK and SIRT1 are important for regulating cocaine reward and dopaminergic (DAergic) activity, with interesting differences depending on whether DAergic activity is in a heightened state and if there is a functional CLOCK protein. Taken together, we find that rhythms in cellular metabolism and circadian proteins work together to regulate dopamine synthesis and the reward value for drugs of abuse.

MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f.sp. tritici infection.

PubMed

Gupta, Om Prakash; Permar, Vipin; Koundal, Vikas; Singh, Uday Dhari; Praveen, Shelly

2012-02-01

Plants have evolved diverse mechanism to recognize pathogen attack and triggers defense responses. These defense responses alter host cellular function regulated by endogenous, small, non-coding miRNAs. To understand the mechanism of miRNAs regulated cellular functions during stem rust infection in wheat, we investigated eight different miRNAs viz. miR159, miR164, miR167, miR171, miR444, miR408, miR1129 and miR1138, involved in three different independent cellular defense response to infection. The investigation reveals that at the initiation of disease, accumulation of miRNAs might be playing a key role in hypersensitive response (HR) from host, which diminishes at the maturation stage. This suggests a possible host-fungal synergistic relation leading to susceptibility. Differential expression of these miRNAs in presence and absence of R gene provides a probable explanation of miRNA regulated R gene mediated independent pathways.

Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium.

PubMed

Delos Santos, Ralph Christian; Bautista, Stephen; Lucarelli, Stefanie; Bone, Leslie N; Dayam, Roya M; Abousawan, John; Botelho, Roberto J; Antonescu, Costin N

2017-10-15

Clathrin-mediated endocytosis is a major regulator of cell-surface protein internalization. Clathrin and other proteins assemble into small invaginating structures at the plasma membrane termed clathrin-coated pits (CCPs) that mediate vesicle formation. In addition, epidermal growth factor receptor (EGFR) signaling is regulated by its accumulation within CCPs. Given the diversity of proteins regulated by clathrin-mediated endocytosis, how this process may distinctly regulate specific receptors is a key question. We examined the selective regulation of clathrin-dependent EGFR signaling and endocytosis. We find that perturbations of phospholipase Cγ1 (PLCγ1), Ca 2+ , or protein kinase C (PKC) impair clathrin-mediated endocytosis of EGFR, the formation of CCPs harboring EGFR, and EGFR signaling. Each of these manipulations was without effect on the clathrin-mediated endocytosis of transferrin receptor (TfR). EGFR and TfR were recruited to largely distinct clathrin structures. In addition to control of initiation and assembly of CCPs, EGF stimulation also elicited a Ca 2+ - and PKC-dependent reduction in synaptojanin1 recruitment to clathrin structures, indicating broad control of CCP assembly by Ca 2+ signals. Hence EGFR elicits PLCγ1-calcium signals to facilitate formation of a subset of CCPs, thus modulating its own signaling and endocytosis. This provides evidence for the versatility of CCPs to control diverse cellular processes. © 2017 Delos Santos et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

PubMed Central

Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy; Groitl, Peter; Wimmer, Peter; Kinkley, Sarah; Mund, Andreas; Everett, Roger D.; Dobner, Thomas

2013-01-01

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling. PMID:23396441

Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases

PubMed Central

Beurel, Eleonore; Grieco, Steven F.; Jope, Richard S.

2014-01-01

Glycogen synthase kinase-3 (GSK3) may be the busiest kinase in most cells, with over 100 known substrates to deal with. How does GSK3 maintain control to selectively phosphorylate each substrate, and why was it evolutionarily favorable for GSK3 to assume such a large responsibility? GSK3 must be particularly adaptable for incorporating new substrates into its repertoire, and we discuss the distinct properties of GSK3 that may contribute to its capacity to fulfill its roles in multiple signaling pathways. The mechanisms regulating GSK3 (predominantly post-translational modifications, substrate priming, cellular trafficking, protein complexes) have been reviewed previously, so here we focus on newly identified complexities in these mechanisms, how each of these regulatory mechanism contributes to the ability of GSK3 to select which substrates to phosphorylate, and how these mechanisms may have contributed to its adaptability as new substrates evolved. The current understanding of the mechanisms regulating GSK3 is reviewed, as are emerging topics in the actions of GSK3, particularly its interactions with receptors and receptor-coupled signal transduction events, and differential actions and regulation of the two GSK3 isoforms, GSK3α and GSK3β. Another remarkable characteristic of GSK3 is its involvement in many prevalent disorders, including psychiatric and neurological diseases, inflammatory diseases, cancer, and others. We address the feasibility of targeting GSK3 therapeutically, and provide an update of its involvement in the etiology and treatment of several disorders. PMID:25435019

New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

PubMed

Broad, W; Ling, Q; Jarvis, P

2016-01-01

Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. Copyright © 2016 Elsevier Inc. All rights reserved.

Caenorhabditis elegans TRPV Channels Function in a Modality-Specific Pathway to Regulate Response to Aberrant Sensory Signaling

PubMed Central

Ezak , Meredith J.; Hong , Elizabeth; Chaparro-Garcia , Angela; Ferkey , Denise M.

2010-01-01

Olfaction and some forms of taste (including bitter) are mediated by G protein-coupled signal transduction pathways. Olfactory and gustatory ligands bind to chemosensory G protein-coupled receptors (GPCRs) in specialized sensory cells to activate intracellular signal transduction cascades. G protein-coupled receptor kinases (GRKs) are negative regulators of signaling that specifically phosphorylate activated GPCRs to terminate signaling. Although loss of GRK function usually results in enhanced cellular signaling, Caenorhabditis elegans lacking GRK-2 function are not hypersensitive to chemosensory stimuli. Instead, grk-2 mutant animals do not chemotax toward attractive olfactory stimuli or avoid aversive tastes and smells. We show here that loss-of-function mutations in the transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 selectively restore grk-2 behavioral avoidance of bitter tastants, revealing modality-specific mechanisms for TRPV channel function in the regulation of C. elegans chemosensation. Additionally, a single amino acid point mutation in OCR-2 that disrupts TRPV channel-mediated gene expression, but does not decrease channel function in chemosensory primary signal transduction, also restores grk-2 bitter taste avoidance. Thus, loss of GRK-2 function may lead to changes in gene expression, via OSM-9/OCR-2, to selectively alter the levels of signaling components that transduce or regulate bitter taste responses. Our results suggest a novel mechanism and multiple modality-specific pathways that sensory cells employ in response to aberrant signal transduction. PMID:20176974

Heat shock cognate 71 (HSC71) regulates cellular antiviral response by impairing formation of VISA aggregates.

PubMed

Liu, Zhigang; Wu, Shu-Wen; Lei, Cao-Qi; Zhou, Qian; Li, Shu; Shu, Hong-Bing; Wang, Yan-Yi

2013-05-01

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.

Evolution and the complexity of bacteriophages.

PubMed

Serwer, Philip

2007-03-13

The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine which, if any, bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamics of homolog evolution. This hypothesis is an explanation of evolutionary leaps in general. If accurate, it will assist both understanding and influencing the evolution of microbes and their communities. Analysis of evolutionary complexity increase for at least prokaryotes should include analysis of genomes of long-genome bacteriophages.

Large-scale Proteomics Analysis of the Human Kinome

PubMed Central

Oppermann, Felix S.; Gnad, Florian; Olsen, Jesper V.; Hornberger, Renate; Greff, Zoltán; Kéri, György; Mann, Matthias; Daub, Henrik

2009-01-01

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis. PMID:19369195

c-di-AMP: An Essential Molecule in the Signaling Pathways that Regulate the Viability and Virulence of Gram-Positive Bacteria

PubMed Central

Fahmi, Tazin; Port, Gary C.

2017-01-01

Signal transduction pathways enable organisms to monitor their external environment and adjust gene regulation to appropriately modify their cellular processes. Second messenger nucleotides including cyclic adenosine monophosphate (c-AMP), cyclic guanosine monophosphate (c-GMP), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) play key roles in many signal transduction pathways used by prokaryotes and/or eukaryotes. Among the various second messenger nucleotides molecules, c-di-AMP was discovered recently and has since been shown to be involved in cell growth, survival, and regulation of virulence, primarily within Gram-positive bacteria. The cellular level of c-di-AMP is maintained by a family of c-di-AMP synthesizing enzymes, diadenylate cyclases (DACs), and degradation enzymes, phosphodiesterases (PDEs). Genetic manipulation of DACs and PDEs have demonstrated that alteration of c-di-AMP levels impacts both growth and virulence of microorganisms. Unlike other second messenger molecules, c-di-AMP is essential for growth in several bacterial species as many basic cellular functions are regulated by c-di-AMP including cell wall maintenance, potassium ion homeostasis, DNA damage repair, etc. c-di-AMP follows a typical second messenger signaling pathway, beginning with binding to receptor molecules to subsequent regulation of downstream cellular processes. While c-di-AMP binds to specific proteins that regulate pathways in bacterial cells, c-di-AMP also binds to regulatory RNA molecules that control potassium ion channel expression in Bacillus subtilis. c-di-AMP signaling also occurs in eukaryotes, as bacterially produced c-di-AMP stimulates host immune responses during infection through binding of innate immune surveillance proteins. Due to its existence in diverse microorganisms, its involvement in crucial cellular activities, and its stimulating activity in host immune responses, c-di-AMP signaling pathway has become an attractive antimicrobial drug target and therefore has been the focus of intensive study in several important pathogens. PMID:28783096

Logic-Based and Cellular Pharmacodynamic Modeling of Bortezomib Responses in U266 Human Myeloma Cells

PubMed Central

Chudasama, Vaishali L.; Ovacik, Meric A.; Abernethy, Darrell R.

2015-01-01

Systems models of biological networks show promise for informing drug target selection/qualification, identifying lead compounds and factors regulating disease progression, rationalizing combinatorial regimens, and explaining sources of intersubject variability and adverse drug reactions. However, most models of biological systems are qualitative and are not easily coupled with dynamical models of drug exposure-response relationships. In this proof-of-concept study, logic-based modeling of signal transduction pathways in U266 multiple myeloma (MM) cells is used to guide the development of a simple dynamical model linking bortezomib exposure to cellular outcomes. Bortezomib is a commonly used first-line agent in MM treatment; however, knowledge of the signal transduction pathways regulating bortezomib-mediated cell cytotoxicity is incomplete. A Boolean network model of 66 nodes was constructed that includes major survival and apoptotic pathways and was updated using responses to several chemical probes. Simulated responses to bortezomib were in good agreement with experimental data, and a reduction algorithm was used to identify key signaling proteins. Bortezomib-mediated apoptosis was not associated with suppression of nuclear factor κB (NFκB) protein inhibition in this cell line, which contradicts a major hypothesis of bortezomib pharmacodynamics. A pharmacodynamic model was developed that included three critical proteins (phospho-NFκB, BclxL, and cleaved poly (ADP ribose) polymerase). Model-fitted protein dynamics and cell proliferation profiles agreed with experimental data, and the model-predicted IC50 (3.5 nM) is comparable to the experimental value (1.5 nM). The cell-based pharmacodynamic model successfully links bortezomib exposure to MM cellular proliferation via protein dynamics, and this model may show utility in exploring bortezomib-based combination regimens. PMID:26163548

Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria.

PubMed

Albert, Lynal S; Brown, Derick G

2015-08-01

In this study we investigated the relationship between a rapid change in extracellular pH and the alteration of bacterial ATP concentration. This relationship is a key component of a hypothesis indicating that bacterial bioenergetics - the creation of ATP from ADP via a proton gradient across the cytoplasmic membrane - can be altered by the physiochemical charge-regulation effect, which results in a pH shift at the bacteria's surface upon adhesion to another surface. The bacterial ATP concentration was measured during a rapid change in extracellular pH from a baseline pH of 7.2 to pH values between 3.5 and 10.5. Experiments were conducted with four neutrophilic bacterial strains, including the Gram-negative Escherichia coli and Pseudomonas putida and the Gram-positive Bacillus subtilis and Staphylococcus epidermidis. A change in bulk pH produced an immediate response in bacterial ATP, demonstrating a direct link between changes in extracellular pH and cellular bioenergetics. In general, the shifts in ATP were similar across the four bacterial strains, with results following an exponential relationship between the extracellular pH and cellular ATP concentration. One exception occurred with S. epidermidis, where there was no variation in cellular ATP at acidic pH values, and this finding is consistent with this species' ability to thrive under acidic conditions. These results provide insight into obtaining a desired bioenergetic response in bacteria through (i) the application of chemical treatments to vary the local pH and (ii) the selection and design of surfaces resulting in local pH modification of attached bacteria via the charge-regulation effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Annexins - scaffolds modulating PKC localization and signaling.

PubMed

Hoque, Monira; Rentero, Carles; Cairns, Rose; Tebar, Francesc; Enrich, Carlos; Grewal, Thomas

2014-06-01

Spatial and temporal organization of signal transduction is critical to link different extracellular stimuli with distinct cellular responses. A classical example of hormones and growth factors creating functional diversity is illustrated by the multiple signaling pathways activated by the protein kinase C (PKC) family of serine/threonine protein kinases. The molecular requirements for diacylglycerol (DAG) and calcium (Ca(2+)) to promote PKC membrane translocation, the hallmark of PKC activation, have been clarified. However, the underlying mechanisms that establish selectivity of individual PKC family members to facilitate differential substrate phosphorylation and varied signal output are still not fully understood. It is now well believed that the coordinated control and functional diversity of PKC signaling involves the formation of PKC isozyme-specific protein complexes in certain subcellular sites. In particular, interaction of PKC isozymes with compartment and signal-organizing scaffolds, including receptors for activated C-kinase (RACKs), A-kinase-anchoring proteins (AKAPs), 14-3-3, heat shock proteins (HSP), and importins target PKC isozymes to specific cellular locations, thereby delivering PKC isozymes into close proximity of their substrates. In addition, several annexins (Anx), including AnxA1, A2, A5 and A6, display specific and distinct abilities to interact and promote membrane targeting of different PKC isozymes. Together with the ability of annexins to create specific membrane microenvironments, this is likely to enable PKCs to phosphorylate certain substrates and regulate their downstream effector pathways in specific cellular sites. This review aims to summarize the capacity of annexins to modulate the localization and activity of PKC family members and participate in the spatiotemporal regulation of PKC signaling in health and disease. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu

Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 humanmore » skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA(III) perturbs Nrf2 pathway and selenoprotein synthesis.« less

Stochastic Nature in Cellular Processes

NASA Astrophysics Data System (ADS)

Liu, Bo; Liu, Sheng-Jun; Wang, Qi; Yan, Shi-Wei; Geng, Yi-Zhao; Sakata, Fumihiko; Gao, Xing-Fa

2011-11-01

The importance of stochasticity in cellular processes is increasingly recognized in both theoretical and experimental studies. General features of stochasticity in gene regulation and expression are briefly reviewed in this article, which include the main experimental phenomena, classification, quantization and regulation of noises. The correlation and transmission of noise in cascade networks are analyzed further and the stochastic simulation methods that can capture effects of intrinsic and extrinsic noise are described.

Regulation of TGF-β signaling, exit from the cell cycle, and cellular migration through cullin cross-regulation: SCF-FBXO11 turns off CRL4-Cdt2.

PubMed

Abbas, Tarek; Keaton, Mignon; Dutta, Anindya

2013-07-15

Deregulation of the cell cycle and genome instability are common features of cancer cells and various mechanisms exist to preserve the integrity of the genome and guard against cancer. The cullin 4-RING ubiquitin ligase (CRL4) with the substrate receptor Cdt2 (CRL4 (Cdt2)) promotes cell cycle progression and prevents genome instability through ubiquitylation and degradation of Cdt1, p21, and Set8 during S phase of the cell cycle and following DNA damage. Two recently published studies report the ubiquitin-dependent degradation of Cdt2 via the cullin 1-RING ubiquitin ligase (CRL1) in association with the substrate specificity factor and tumor suppressor FBXO11 (CRL1 (FBXO11)). The newly identified pathway restrains the activity of CRL4 (Cdt2) on p21 and Set8 and regulates cellular response to TGF-β, exit from the cell cycle and cellular migration. Here, we show that the CRL1 (FBXO11) also promotes the degradation of Cdt2 during an unperturbed cell cycle to promote efficient progression through S and G 2/M phases of the cell cycle. We discuss how this new method of regulating the abundance of Cdt2 participates in various cellular activities.

The Role of Nutraceuticals in Pancreatic Cancer Prevention and Therapy: Targeting Cellular Signaling, MicroRNAs, and Epigenome.

PubMed

Li, Yiwei; Go, Vay Liang W; Sarkar, Fazlul H

2015-01-01

Pancreatic cancer is one of the most aggressive malignancies in US adults. Experimental studies have found that antioxidant nutrients could reduce oxidative DNA damage, suggesting that these antioxidants may protect against pancreatic carcinogenesis. Several epidemiologic studies showed that dietary intake of antioxidants was inversely associated with the risk for pancreatic cancer, demonstrating the inhibitory effects of antioxidants on pancreatic carcinogenesis. Moreover, nutraceuticals, the anticancer agents from diet or natural plants, have been found to inhibit the development and progression of pancreatic cancer through the regulation of cellular signaling pathways. Importantly, nutraceuticals also up-regulate the expression of tumor-suppressive microRNAs (miRNAs) and down-regulate the expression of oncogenic miRNAs, leading to the inhibition of pancreatic cancer cell growth and pancreatic cancer stem cell self-renewal through modulation of cellular signaling network. Furthermore, nutraceuticals also regulate epigenetically deregulated DNAs and miRNAs, leading to the normalization of altered cellular signaling in pancreatic cancer cells. Therefore, nutraceuticals could have much broader use in the prevention and/or treatment of pancreatic cancer in combination with conventional chemotherapeutics. However, more in vitro mechanistic experiments, in vivo animal studies, and clinical trials are needed to realize the true value of nutraceuticals in the prevention and/or treatment of pancreatic cancer.

The role of nutraceuticals in pancreatic cancer prevention and therapy: Targeting cellular signaling, miRNAs and epigenome

PubMed Central

Li, Yiwei; Go, Vay Liang W.; Sarkar, Fazlul H.

2014-01-01

Pancreatic cancer is one of the most aggressive malignancies in US adults. The experimental studies have found that antioxidant nutrients could reduce oxidative DNA damage, suggesting that these antioxidants may protect against pancreatic carcinogenesis. Several epidemiologic studies showed that dietary intake of antioxidants was inversely associated with the risk of pancreatic cancer, demonstrating the inhibitory effects of antioxidants on pancreatic carcinogenesis. Moreover, nutraceuticals, the anti-cancer agents from diet or natural plants, have been found to inhibit the development and progression of pancreatic cancer through the regulation of cellular signaling pathways. Importantly, nutraceuticals also up-regulate the expression of tumor suppressive miRNAs and down-regulate the expression of oncogenic miRNAs, leading to the inhibition of pancreatic cancer cell growth and pancreatic Cancer Stem Cell (CSC) self-renewal through modulation of cellular signaling network. Furthermore, nutraceuticals also regulate epigenetically deregulated DNAs and miRNAs, leading to the normalization of altered cellular signaling in pancreatic cancer cells. Therefore, nutraceuticals could have much broader use in the prevention and/or treatment of pancreatic cancer in combination with conventional chemotherapeutics. However, more in vitro mechanistic experiments, in vivo animal studies, and clinical trials are needed to realize the true value of nutraceuticals in the prevention and/or treatment of pancreatic cancer. PMID:25493373

Epigenetic regulation of cell fate reprogramming in aging and disease: A predictive computational model.

PubMed

Folguera-Blasco, Núria; Cuyàs, Elisabet; Menéndez, Javier A; Alarcón, Tomás

2018-03-01

Understanding the control of epigenetic regulation is key to explain and modify the aging process. Because histone-modifying enzymes are sensitive to shifts in availability of cofactors (e.g. metabolites), cellular epigenetic states may be tied to changing conditions associated with cofactor variability. The aim of this study is to analyse the relationships between cofactor fluctuations, epigenetic landscapes, and cell state transitions. Using Approximate Bayesian Computation, we generate an ensemble of epigenetic regulation (ER) systems whose heterogeneity reflects variability in cofactor pools used by histone modifiers. The heterogeneity of epigenetic metabolites, which operates as regulator of the kinetic parameters promoting/preventing histone modifications, stochastically drives phenotypic variability. The ensemble of ER configurations reveals the occurrence of distinct epi-states within the ensemble. Whereas resilient states maintain large epigenetic barriers refractory to reprogramming cellular identity, plastic states lower these barriers, and increase the sensitivity to reprogramming. Moreover, fine-tuning of cofactor levels redirects plastic epigenetic states to re-enter epigenetic resilience, and vice versa. Our ensemble model agrees with a model of metabolism-responsive loss of epigenetic resilience as a cellular aging mechanism. Our findings support the notion that cellular aging, and its reversal, might result from stochastic translation of metabolic inputs into resilient/plastic cell states via ER systems.

Prostaglandin E₂ regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells.

PubMed

Fujino, Hiromichi; Toyomura, Kaori; Chen, Xiao-bo; Regan, John W; Murayama, Toshihiko

2011-02-01

An important event in the development of tumors is angiogenesis, or the formation of new blood vessels. Angiogenesis is also known to be involved in tumor cell metastasis and is dependent upon the activity of the vascular endothelial growth factor (VEGF) signaling pathway. Studies of mice in which the EP3 prostanoid receptors have been genetically deleted have shown a role for these receptors in cancer growth and angiogenesis. In the present study, human colon cancer HCA-7 cells were used as a model system to understand the potential role of EP3 receptors in tumor cell migration. We now show that stimulation of HCA-7 cells with PGE₂ enhanced the up-regulation of VEGF receptor-1 (VEGFR-1) expression by a mechanism involving EP3 receptor-mediated activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Moreover, the PGE₂ stimulated increase in VEGFR-1 expression was accompanied by an increase in the cellular migration of HCA-7 cells. Given the known involvement of VEGFR-1 in cellular migration, our results suggest that EP3 receptors may contribute to tumor cell metastasis by increasing cellular migration through the up-regulation of VEGFR-1 signaling. Copyright © 2010 Elsevier Inc. All rights reserved.

Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

PubMed Central

Ding, Yan; Liu, Zixing; Desai, Shruti; Zhao, Yuhua; Liu, Hao; Pannell, Lewis K; Yi, Hong; Wright, Elizabeth R; Owen, Laurie B; Dean-Colomb, Windy; Fodstad, Oystein; Lu, Jianrong; LeDoux, Susan P; Wilson, Glenn L; Tan, Ming

2012-01-01

It is well known that ErbB2, a receptor tyrosine kinase, localizes on the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through the association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to ErbB2 targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. PMID:23232401

Cell Proliferation, Reactive Oxygen and Cellular Glutathione

PubMed Central

Day, Regina M.; Suzuki, Yuichiro J.

2005-01-01

A variety of cellular activities, including metabolism, growth, and death, are regulated and modulated by the redox status of the environment. A biphasic effect has been demonstrated on cellular proliferation with reactive oxygen species (ROS)—especially hydrogen peroxide and superoxide—in which low levels (usually submicromolar concentrations) induce growth but higher concentrations (usually >10–30 micromolar) induce apoptosis or necrosis. This phenomenon has been demonstrated for primary, immortalized and transformed cell types. However, the mechanism of the proliferative response to low levels of ROS is not well understood. Much of the work examining the signal transduction by ROS, including H2O2, has been performed using doses in the lethal range. Although use of higher ROS doses have allowed the identification of important signal transduction pathways, these pathways may be activated by cells only in association with ROS-induced apoptosis and necrosis, and may not utilize the same pathways activated by lower doses of ROS associated with increased cell growth. Recent data has shown that low levels of exogenous H2O2 up-regulate intracellular glutathione and activate the DNA binding activity toward antioxidant response element. The modulation of the cellular redox environment, through the regulation of cellular glutathione levels, may be a part of the hormetic effect shown by ROS on cell growth. PMID:18648617

Selective Sensitization of Zinc Finger Protein Oxidation by Reactive Oxygen Species through Arsenic Binding*

PubMed Central

Zhou, Xixi; Cooper, Karen L.; Sun, Xi; Liu, Ke J.; Hudson, Laurie G.

2015-01-01

Cysteine oxidation induced by reactive oxygen species (ROS) on redox-sensitive targets such as zinc finger proteins plays a critical role in redox signaling and subsequent biological outcomes. We found that arsenic exposure led to oxidation of certain zinc finger proteins based on arsenic interaction with zinc finger motifs. Analysis of zinc finger proteins isolated from arsenic-exposed cells and zinc finger peptides by mass spectrometry demonstrated preferential oxidation of C3H1 and C4 zinc finger configurations. C2H2 zinc finger proteins that do not bind arsenic were not oxidized by arsenic-generated ROS in the cellular environment. The findings suggest that selectivity in arsenic binding to zinc fingers with three or more cysteines defines the target proteins for oxidation by ROS. This represents a novel mechanism of selective protein oxidation and demonstrates how an environmental factor may sensitize certain target proteins for oxidation, thus altering the oxidation profile and redox regulation. PMID:26063799

The mystery of membrane organization: composition, regulation and physiological relevance of lipid rafts

PubMed Central

Sezgin, Erdinc; Levental, Ilya; Mayor, Satyajit; Eggeling, Christian

2017-01-01

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large body of research has focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, wherein the preferential associations of cholesterol and saturated lipids drives the formation of relatively packed (ordered) membrane domains that selectively recruit certain lipids and proteins. Recent years have yielded new insights into this concept and its in vivo relevance, primarily owing to the development of biochemical and biophysical technologies. PMID:28356571

Dynamic Palmitoylation and the Role of DHHC Proteins in T Cell Activation and Anergy

PubMed Central

Ladygina, Nadejda; Martin, Brent R.; Altman, Amnon

2017-01-01

Although protein S-palmitoylation was first characterized >30 years ago, and is implicated in the function, trafficking, and localization of many proteins, little is known about the regulation and physiological implications of this posttranslational modification. Palmitoylation of various signaling proteins required for TCR-induced T cell activation is also necessary for their proper function. LAT (linker for activation of T cells) is an essential scaffolding protein involved in T cell development and activation, and we found that its palmitoylation is selectively impaired in anergic T cells. The recent discovery of the DHHC family of palmitoyl acyl transferases (PATs) and the establishment of sensitive and quantitative proteomics-based methods for global analysis of the palmitoyl proteome led to significant progress in studying the biology and underlying mechanisms of cellular protein palmitoylation. We are using these approaches to explore the palmitoyl proteome in T lymphocytes and, specifically, the mechanistic basis for the impaired palmitoylation of LAT in anergic T cells. This chapter reviews the history of protein palmitoylation and its role in T cell activation, the DHHC family and new methodologies for global analysis of the palmitoyl proteome, and summarizes our recent work in this area. The new methodologies will accelerate the pace of research and provide a greatly improved mechanistic and molecular understanding of the complex process of protein palmitoylation and its regulation, and the substrate specificity of the novel DHHC family. Reversible protein palmitoylation will likely prove to be an important posttranslational mechanism that regulates cellular responses, similar to protein phosphorylation and ubiquitination. PMID:21569911

Selective Regulation of Human Immunodeficiency Virus-Infected CD4+ Lymphocytes by a Synthetic Immunomodulator Leads to Potent Virus Suppression In Vitro and in hu-PBL-SCID Mice

PubMed Central

Bahr, George M.; Darcissac, Edith C. A.; Castéran, Nathalie; Amiel, Corinne; Cocude, Cécile; Truong, Marie-José; Dewulf, Joëlle; Capron, André; Mouton, Yves

2001-01-01

We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with β-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the host's nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication. PMID:11435574

Genome-wide analysis of drought induced gene expression changes in flax (Linum usitatissimum).

PubMed

Dash, Prasanta K; Cao, Yongguo; Jailani, Abdul K; Gupta, Payal; Venglat, Prakash; Xiang, Daoquan; Rai, Rhitu; Sharma, Rinku; Thirunavukkarasu, Nepolean; Abdin, Malik Z; Yadava, Devendra K; Singh, Nagendra K; Singh, Jas; Selvaraj, Gopalan; Deyholos, Mike; Kumar, Polumetla Ananda; Datla, Raju

2014-01-01

A robust phenotypic plasticity to ward off adverse environmental conditions determines performance and productivity in crop plants. Flax (linseed), is an important cash crop produced for natural textile fiber (linen) or oilseed with many health promoting products. This crop is prone to drought stress and yield losses in many parts of the world. Despite recent advances in drought research in a number of important crops, related progress in flax is very limited. Since, response of this plant to drought stress has not been addressed at the molecular level; we conducted microarray analysis to capture transcriptome associated with induced drought in flax. This study identified 183 differentially expressed genes (DEGs) associated with diverse cellular, biophysical and metabolic programs in flax. The analysis also revealed especially the altered regulation of cellular and metabolic pathways governing photosynthesis. Additionally, comparative transcriptome analysis identified a plethora of genes that displayed differential regulation both spatially and temporally. These results revealed co-regulated expression of 26 genes in both shoot and root tissues with implications for drought stress response. Furthermore, the data also showed that more genes are upregulated in roots compared to shoots, suggesting that roots may play important and additional roles in response to drought in flax. With prolonged drought treatment, the number of DEGs increased in both tissue types. Differential expression of selected genes was confirmed by qRT-PCR, thus supporting the suggested functional association of these intrinsic genes in maintaining growth and homeostasis in response to imminent drought stress in flax. Together the present study has developed foundational and new transcriptome data sets for drought stress in flax.

A small molecule inhibitor of Rheb selectively targets mTORC1 signaling.

PubMed

Mahoney, Sarah J; Narayan, Sridhar; Molz, Lisa; Berstler, Lauren A; Kang, Seong A; Vlasuk, George P; Saiah, Eddine

2018-02-07

The small G-protein Rheb activates the mechanistic target of rapamycin complex 1 (mTORC1) in response to growth factor signals. mTORC1 is a master regulator of cellular growth and metabolism; aberrant mTORC1 signaling is associated with fibrotic, metabolic, and neurodegenerative diseases, cancers, and rare disorders. Point mutations in the Rheb switch II domain impair its ability to activate mTORC1. Here, we report the discovery of a small molecule (NR1) that binds Rheb in the switch II domain and selectively blocks mTORC1 signaling. NR1 potently inhibits mTORC1 driven phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) but does not inhibit phosphorylation of AKT or ERK. In contrast to rapamycin, NR1 does not cause inhibition of mTORC2 upon prolonged treatment. Furthermore, NR1 potently and selectively inhibits mTORC1 in mouse kidney and muscle in vivo. The data presented herein suggest that pharmacological inhibition of Rheb is an effective approach for selective inhibition of mTORC1 with therapeutic potential.

Serotonin-related pathways and developmental plasticity: relevance for psychiatric disorders

PubMed Central

Dayer, Alexandre

2014-01-01

Risk for adult psychiatric disorders is partially determined by early-life alterations occurring during neural circuit formation and maturation. In this perspective, recent data show that the serotonin system regulates key cellular processes involved in the construction of cortical circuits. Translational data for rodents indicate that early-life serotonin dysregulation leads to a wide range of behavioral alterations, ranging from stress-related phenotypes to social deficits. Studies in humans have revealed that serotonin-related genetic variants interact with early-life stress to regulate stress-induced cortisol responsiveness and activate the neural circuits involved in mood and anxiety disorders. Emerging data demonstrate that early-life adversity induces epigenetic modifications in serotonin-related genes. Finally, recent findings reveal that selective serotonin reuptake inhibitors can reinstate juvenile-like forms of neural plasticity, thus allowing the erasure of long-lasting fear memories. These approaches are providing new insights on the biological mechanisms and clinical application of antidepressants. PMID:24733969

Mineralocorticoid regulation of cell function: the role of rapid signalling and gene transcription pathways.

PubMed

Ong, Gregory S Y; Young, Morag J

2017-01-01

The mineralocorticoid receptor (MR) and mineralocorticoids regulate epithelial handling of electrolytes, and induces diverse effects on other tissues. Traditionally, the effects of MR were ascribed to ligand-receptor binding and activation of gene transcription. However, the MR also utilises a number of intracellular signalling cascades, often by transactivating unrelated receptors, to change cell function more rapidly. Although aldosterone is the physiological mineralocorticoid, it is not the sole ligand for MR. Tissue-selective and mineralocorticoid-specific effects are conferred through the enzyme 11β-hydroxysteroid dehydrogenase 2, cellular redox status and properties of the MR itself. Furthermore, not all aldosterone effects are mediated via MR, with implication of the involvement of other membrane-bound receptors such as GPER. This review will describe the ligands, receptors and intracellular mechanisms available for mineralocorticoid hormone and receptor signalling and illustrate their complex interactions in physiology and disease. © 2017 Society for Endocrinology.

Epigenetic mechanisms in memory and synaptic function

PubMed Central

Sultan, Faraz A; Day, Jeremy J

2011-01-01

Although the term ‘epigenetics’ was coined nearly seventy years ago, its critical function in memory processing by the adult CNS has only recently been appreciated. The hypothesis that epigenetic mechanisms regulate memory and behavior was motivated by the need for stable molecular processes that evade turnover of the neuronal proteome. In this article, we discuss evidence that supports a role for neural epigenetic modifications in the formation, consolidation and storage of memory. In addition, we will review the evidence that epigenetic mechanisms regulate synaptic plasticity, a cellular correlate of memory. We will also examine how the concerted action of multiple epigenetic mechanisms with varying spatiotemporal profiles influence selective gene expression in response to behavioral experience. Finally, we will suggest key areas for future research that will help elucidate the complex, vital and still mysterious, role of epigenetic mechanisms in neural function and behavior. PMID:22122279

Estrogen modulation properties of mangiferin and quercetin and the mangiferin metabolite norathyriol.

PubMed

Wilkinson, Ashley S; Taing, Meng-Wong; Pierson, Jean Thomas; Lin, Chun-Nam; Dietzgen, Ralf G; Shaw, P Nicholas; Gidley, Michael J; Monteith, Gregory R; Roberts-Thomson, Sarah J

2015-06-01

Mango fruit contain many bioactive compounds, some of which are transcription factor regulators. Estrogen receptor alpha (ERα) and beta (ERβ) are two regulators of gene transcription that are important in a variety of physiological processes and also in diseases including breast cancer. We examined the ability of the mango constituents quercetin, mangiferin, and the aglycone form of mangiferin, norathyriol, to activate both isoforms of the estrogen receptor. Quercetin and norathyriol decreased the viability of MCF-7 breast cancer cells whereas mangiferin had no effect on MCF-7 cells. We also determined that quercetin and mangiferin selectively activated ERα whereas norathyriol activated both ERα and ERβ. Despite quercetin, mangiferin and norathyriol having similar polyphenolic structural motifs, only norathyriol activated ERβ, showing that bioactive agents in mangoes have very specific biological effects. Such specificity may be important given the often-opposing roles of ERα and ERβ in breast cancer proliferation and other cellular processes.

Avian W and mammalian Y chromosomes convergently retained dosage-sensitive regulators

PubMed Central

Bellott, Daniel W.; Skaletsky, Helen; Cho, Ting-Jan; Brown, Laura; Locke, Devin; Chen, Nancy; Galkina, Svetlana; Pyntikova, Tatyana; Koutseva, Natalia; Graves, Tina; Kremitzki, Colin; Warren, Wesley C.; Clark, Andrew G.; Gaginskaya, Elena; Wilson, Richard K.; Page, David C.

2017-01-01

After birds diverged from mammals, different ancestral autosomes evolved into sex chromosomes in each lineage. In birds, females are ZW and males ZZ, but in mammals females are XX and males XY. We sequenced the chicken W chromosome, compared its gene content with our reconstruction of the ancestral autosomes, and followed the evolutionary trajectory of ancestral W-linked genes across birds. Avian W chromosomes evolved in parallel with mammalian Y chromosomes, preserving ancestral genes through selection to maintain the dosage of broadly-expressed regulators of key cellular processes. We propose that, like the human Y chromosome, the chicken W chromosome is essential for embryonic viability of the heterogametic sex. Unlike other sequenced sex chromosomes, the chicken W did not acquire and amplify genes specifically expressed in reproductive tissues. We speculate that the pressures that drive the acquisition of reproduction related genes on sex chromosomes may be specific to the male germ line. PMID:28135246

Regulation of cell function by methionine oxidation and reduction

PubMed Central

Hoshi, Toshinori; Heinemann, Stefan H

2001-01-01

Reactive oxygen species (ROS) are generated during normal cellular activity and may exist in excess in some pathophysiological conditions, such as inflammation or reperfusion injury. These molecules oxidize a variety of cellular constituents, but sulfur-containing amino acid residues are especially susceptible. While reversible cysteine oxidation and reduction is part of well-established signalling systems, the oxidation and the enzymatically catalysed reduction of methionine is just emerging as a novel molecular mechanism for cellular regulation. Here we discuss how the oxidation of methionine to methionine sulfoxide in signalling proteins such as ion channels affects the function of these target proteins. Methionine sulfoxide reductase, which reduces methionine sulfoxide to methionine in a thioredoxin-dependent manner, is therefore not only an enzyme important for the repair of age- or degenerative disease-related protein modifications. It is also a potential missing link in the post-translational modification cycle involved in the specific oxidation and reduction of methionine residues in cellular signalling proteins, which may give rise to activity-dependent plastic changes in cellular excitability. PMID:11179387

Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.

PubMed

Edmonds, Matthew J; Carter, Rachel J; Nickson, Catherine M; Williams, Sarah C; Parsons, Jason L

2017-01-25

Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

PubMed Central

Sahara, Hiroeki; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-01-01

Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology. PMID:21559518

Pore dilatation increases the bicarbonate permeability of CFTR, ANO1 and glycine receptor anion channels

PubMed Central

Jun, Ikhyun; Cheng, Mary Hongying; Sim, Eunji; Jung, Jinsei; Suh, Bong Lim; Kim, Yonjung; Son, Hankil; Park, Kyungsoo; Kim, Chul Hoon; Yoon, Joo‐Heon; Whitcomb, David C.; Bahar, Ivet

2016-01-01

Key points Cellular stimuli can modulate the ion selectivity of some anion channels, such as CFTR, ANO1 and the glycine receptor (GlyR), by changing pore size.Ion selectivity of CFTR, ANO1 and GlyR is critically affected by the electric permittivity and diameter of the channel pore.Pore size change affects the energy barriers of ion dehydration as well as that of size‐exclusion of anion permeation.Pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of CFTR, ANO1 and GlyR.Dynamic change in P HC O3/ Cl may mediate many physiological and pathological processes. Abstract Chloride (Cl−) and bicarbonate (HCO3 −) are two major anions and their permeation through anion channels plays essential roles in our body. However, the mechanism of ion selection by the anion channels is largely unknown. Here, we provide evidence that pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of anion channels by reducing energy barriers of size‐exclusion and ion dehydration of HCO3 − permeation. Molecular, physiological and computational analyses of major anion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR), anoctamin‐1(ANO1/TMEM16A) and the glycine receptor (GlyR), revealed that the ion selectivity of anion channels is basically determined by the electric permittivity and diameter of the pore. Importantly, cellular stimuli dynamically modulate the anion selectivity of CFTR and ANO1 by changing the pore size. In addition, pore dilatation by a mutation in the pore‐lining region alters the anion selectivity of GlyR. Changes in pore size affected not only the energy barriers of size exclusion but that of ion dehydration by altering the electric permittivity of water‐filled cavity in the pore. The dynamic increase in P HC O3/ Cl by pore dilatation may have many physiological and pathophysiological implications ranging from epithelial HCO3 − secretion to neuronal excitation. PMID:26663196

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.

PubMed

Smith, Jeffrey S; Rajagopal, Sudarshan

2016-04-22

The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of µ-Opioid Receptors: Desensitization, Phosphorylation, Internalization, and Tolerance

PubMed Central

Williams, John T.; Ingram, Susan L.; Henderson, Graeme; Chavkin, Charles; von Zastrow, Mark; Schulz, Stefan; Koch, Thomas; Evans, Christopher J.

2013-01-01

Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance. PMID:23321159

Improved intra-array and interarray normalization of peptide microarray phosphorylation for phosphorylome and kinome profiling by rational selection of relevant spots

PubMed Central

Scholma, Jetse; Fuhler, Gwenny M.; Joore, Jos; Hulsman, Marc; Schivo, Stefano; List, Alan F.; Reinders, Marcel J. T.; Peppelenbosch, Maikel P.; Post, Janine N.

2016-01-01

Massive parallel analysis using array technology has become the mainstay for analysis of genomes and transcriptomes. Analogously, the predominance of phosphorylation as a regulator of cellular metabolism has fostered the development of peptide arrays of kinase consensus substrates that allow the charting of cellular phosphorylation events (often called kinome profiling). However, whereas the bioinformatical framework for expression array analysis is well-developed, no advanced analysis tools are yet available for kinome profiling. Especially intra-array and interarray normalization of peptide array phosphorylation remain problematic, due to the absence of “housekeeping” kinases and the obvious fallacy of the assumption that different experimental conditions should exhibit equal amounts of kinase activity. Here we describe the development of analysis tools that reliably quantify phosphorylation of peptide arrays and that allow normalization of the signals obtained. We provide a method for intraslide gradient correction and spot quality control. We describe a novel interarray normalization procedure, named repetitive signal enhancement, RSE, which provides a mathematical approach to limit the false negative results occuring with the use of other normalization procedures. Using in silico and biological experiments we show that employing such protocols yields superior insight into cellular physiology as compared to classical analysis tools for kinome profiling. PMID:27225531

Reactive Neuroblastosis in Huntington’s Disease: A Putative Therapeutic Target for Striatal Regeneration in the Adult Brain

PubMed Central

Kandasamy, Mahesh; Aigner, Ludwig

2018-01-01

The cellular and molecular mechanisms underlying the reciprocal relationship between adult neurogenesis, cognitive and motor functions have been an important focus of investigation in the establishment of effective neural replacement therapies for neurodegenerative disorders. While neuronal loss, reactive gliosis and defects in the self-repair capacity have extensively been characterized in neurodegenerative disorders, the transient excess production of neuroblasts detected in the adult striatum of animal models of Huntington’s disease (HD) and in post-mortem brain of HD patients, has only marginally been addressed. This abnormal cellular response in the striatum appears to originate from the selective proliferation and ectopic migration of neuroblasts derived from the subventricular zone (SVZ). Based on and in line with the term “reactive astrogliosis”, we propose to name the observed cellular event “reactive neuroblastosis”. Although, the functional relevance of reactive neuroblastosis is unknown, we speculate that this process may provide support for the tissue regeneration in compensating the structural and physiological functions of the striatum in lieu of aging or of the neurodegenerative process. Thus, in this review article, we comprehend different possibilities for the regulation of striatal neurogenesis, neuroblastosis and their functional relevance in the context of HD. PMID:29593498

Regulating Biocompatibility of Carbon Spheres via Defined Nanoscale Chemistry and a Careful Selection of Surface Functionalities

NASA Astrophysics Data System (ADS)

Misra, Santosh K.; Chang, Huei-Huei; Mukherjee, Prabuddha; Tiwari, Saumya; Ohoka, Ayako; Pan, Dipanjan

2015-10-01

A plethora of nanoarchitectures have been evaluated preclincially for applications in early detection and treatment of diseases at molecular and cellular levels resulted in limited success of their clinical translation. It is important to identify the factors that directly or indirectly affect their use in human. We bring a fundamental understanding of how to adjust the biocompatibility of carbon based spherical nanoparticles (CNPs) through defined chemistry and a vigilant choice of surface functionalities. CNPs of various size are designed by tweaking size (2-250 nm), surface chemistries (positive, or negatively charged), molecular chemistries (linear, dendritic, hyperbranched) and the molecular weight of the coating agents (MW 400-20 kDa). A combination of in vitro assays as tools were performed to determine the critical parameters that may trigger toxicity. Results indicated that hydrodynamic sizes are potentially not a risk factor for triggering cellular and systemic toxicity, whereas the presence of a highly positive surface charge and increasing molecular weight enhance the chance of inducing complement activation. Bare and carboxyl-terminated CNPs did present some toxicity at the cellular level which, however, is not comparable to those caused by positively charged CNPs. Similarly, negatively charged CNPs with hydroxyl and carboxylic functionalities did not cause any hemolysis.

Airway smooth muscle contraction - perspectives on past, present and future.

PubMed

Mitchell, H W

2009-10-01

Past and contemporary views of airway smooth muscle (ASM) have led to a high level of understanding of the control and intracellular regulation of force or shortening of ASM and of its possible role in airway disease. As well as the multitude of cellular mechanisms that regulate ASM contraction, a number of structural and mechanical factors, which are only present at the airway and lung level, provide overriding control over ASM. With new knowledge about the cellular physiology and biology of ASM, there is increasing need to understand how ASM contraction is regulated and expressed at these airway and system levels.

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Oxidative Stress, Bone Marrow Failure, and Genome Instability in Hematopoietic Stem Cells

PubMed Central

Richardson, Christine; Yan, Shan; Vestal, C. Greer

2015-01-01

Reactive oxygen species (ROS) can be generated by defective endogenous reduction of oxygen by cellular enzymes or in the mitochondrial respiratory pathway, as well as by exogenous exposure to UV or environmental damaging agents. Regulation of intracellular ROS levels is critical since increases above normal concentrations lead to oxidative stress and DNA damage. A growing body of evidence indicates that the inability to regulate high levels of ROS leading to alteration of cellular homeostasis or defective repair of ROS-induced damage lies at the root of diseases characterized by both neurodegeneration and bone marrow failure as well as cancer. That these diseases may be reflective of the dynamic ability of cells to respond to ROS through developmental stages and aging lies in the similarities between phenotypes at the cellular level. This review summarizes work linking the ability to regulate intracellular ROS to the hematopoietic stem cell phenotype, aging, and disease. PMID:25622253

Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.

PubMed

Lapek, John D; Greninger, Patricia; Morris, Robert; Amzallag, Arnaud; Pruteanu-Malinici, Iulian; Benes, Cyril H; Haas, Wilhelm

2017-10-01

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

PubMed Central

Walley, Justin W.; Rowe, Heather C.; Xiao, Yanmei; Chehab, E. Wassim; Kliebenstein, Daniel J.; Wagner, Doris; Dehesh, Katayoon

2008-01-01

Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks. PMID:19079584

The chromatin remodeler SPLAYED regulates specific stress signaling pathways.

PubMed

Walley, Justin W; Rowe, Heather C; Xiao, Yanmei; Chehab, E Wassim; Kliebenstein, Daniel J; Wagner, Doris; Dehesh, Katayoon

2008-12-01

Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks.

Antagonism of dopamine D2 receptors alters phosphorylation of Fyn in the rat medial prefrontal cortex

PubMed Central

Mao, Li-Min; Wang, John Q.

2017-01-01

Several Src family kinase (SFK) members are expressed in the mammalian brain and serve as key kinases in the regulation of a variety of cellular and synaptic events. These SFKs may be subject to the modulation by dopamine, although this topic has been investigated incompletely. In this study, we explored whether dopamine D2 receptors (D2R) regulate SFKs in adult rat brains in vivo. We investigated the role of D2Rs in two forebrain areas, the medial prefrontal cortex (mPFC) and hippocampus, since dopamine plays a pivotal role in regulating activity of mPFC and hippocampal neurons and D2Rs are expressed in these regions. We found that a systemic injection of a D2R selective antagonist eticlopride elevated phosphorylation of SFKs at a conserved autophosphorylation site, an event correlated with activation of SFKs, in the mPFC. Similarly, antagonism of D2Rs by haloperidol increased SFK phosphorylation. In contrast, eticlopride and haloperidol did not alter SFK phosphorylation in the hippocampus. The effect of eticlopride was time-dependent and relatively delayed. Among two common SFK members enriched at synaptic sites, eticlopride selectively altered phosphorylation of Fyn but not Src. Our data suggest that D2Rs exert an inhibitory effect on the activity-related phosphorylation of Fyn in the mPFC under normal conditions. PMID:28176147

Xenophagic pathways and their bacterial subversion in cellular self-defense - παντα ρει - everything is in flux.

PubMed

Radomski, Nadine; Rebbig, Annica; Leonhardt, Ralf M; Knittler, Michael R

2017-11-02

Autophagy is an evolutionarily ancient and highly conserved eukaryotic mechanism that targets cytoplasmic material for degradation. Autophagic flux involves the formation of autophagosomes and their degradation by lysosomes. The process plays a crucial role in maintaining cellular homeostasis and responds to various environmental conditions. While autophagy had previously been thought to be a non-selective process, it is now clear that it can also selectively target cellular organelles, such as mitochondria (referred to as mitophagy) and/or invading pathogens (referred to as xenophagy). Selective autophagy is characterized by specific substrate recognition and requires distinct cellular adaptor proteins. Here we review xenophagic mechanisms involved in the recognition and autolysosomal or autophagolysosomal degradation of different intracellular bacteria. In this context, we also discuss a recently discovered cellular self-defense pathway, termed mito-xenophagy, which occurs during bacterial infection of dendritic cells and depends on a TNF-α-mediated metabolic switch from oxidative phosphorylation to glycolysis. Copyright © 2017 Elsevier GmbH. All rights reserved.

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters.

PubMed

Du, Wei; Rani, Reena; Sipple, Jared; Schick, Jonathan; Myers, Kasiani C; Mehta, Parinda; Andreassen, Paul R; Davies, Stella M; Pang, Qishen

2012-05-03

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Specificity and autoregulation of Notch binding by tandem WW domains in suppressor of Deltex.

PubMed

Jennings, Martin D; Blankley, Richard T; Baron, Martin; Golovanov, Alexander P; Avis, Johanna M

2007-09-28

WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of σ-factor activity by proteolysis

PubMed Central

Jaiswal, Ravi K.; Prabha, Tangirala Surya; Manjeera, Gowravaram; Gopal, Balasubramanian

2013-01-01

The relative levels of different σ factors dictate the expression profile of a bacterium. Extracytoplasmic function σ factors synchronize the transcriptional profile with environmental conditions. The cellular concentration of free extracytoplasmic function σ factors is regulated by the localization of this protein in a σ/anti-σ complex. Anti-σ factors are multi-domain proteins with a receptor to sense environmental stimuli and a conserved anti-σ domain (ASD) that binds a σ factor. Here we describe the structure of Mycobacterium tuberculosis anti-σD (RsdA) in complex with the -35 promoter binding domain of σD (σD4). We note distinct conformational features that enable the release of σD by the selective proteolysis of the ASD in RsdA. The structural and biochemical features of the σD/RsdA complex provide a basis to reconcile diverse regulatory mechanisms that govern σ/anti-σ interactions despite high overall structural similarity. Multiple regulatory mechanisms embedded in an ASD scaffold thus provide an elegant route to rapidly re-engineer the expression profile of a bacterium in response to an environmental stimulus. PMID:23314154

GDC-0941, a novel class I selective PI3K inhibitor, enhances the efficacy of docetaxel in human breast cancer models by increasing cell death in vitro and in vivo.

PubMed

Wallin, Jeffrey J; Guan, Jane; Prior, Wei Wei; Lee, Leslie B; Berry, Leanne; Belmont, Lisa D; Koeppen, Hartmut; Belvin, Marcia; Friedman, Lori S; Sampath, Deepak

2012-07-15

Docetaxel is a front-line standard-of-care chemotherapeutic drug for the treatment of breast cancer. Phosphoinositide 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, migration, and survival. The current study was intended to determine whether GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the antitumor activity of docetaxel in human breast cancer models in vitro and in vivo. A panel of 25 breast tumor cell lines representing HER2+, luminal, and basal subtypes were treated with GDC-0941, docetaxel, or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers, and apoptosis induction. Drug combination effects on cellular viability were also assessed in nontransformed MCF10A human mammary epithelial cells. Human xenografts of breast cancer cell lines and patient-derived tumors were used to assess efficacy of GDC-0941 and docetaxel in vivo. Combination of GDC-0941 and docetaxel decreased the cellular viability of breast tumor cell lines in vitro but to variable degrees of drug synergy. Compared with nontransformed MCF10A cells, the addition of both drugs resulted in stronger synergistic effects in a subset of tumor cell lines that were not predicted by breast cancer subtype. In xenograft models, GDC-0941 enhanced the antitumor activity of docetaxel with maximum combination efficacy observed within 1 hour of administering both drugs. GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon cotreatment with docetaxel. GDC-0941 augments the efficacy of docetaxel by increasing drug-induced apoptosis in breast cancer models.

Expression of an Engineered Heterologous Antimicrobial Peptide in Potato Alters Plant Development and Mitigates Normal Abiotic and Biotic Responses

PubMed Central

Goyal, Ravinder K.; Hancock, Robert E. W.; Mattoo, Autar K.; Misra, Santosh

2013-01-01

Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield. PMID:24147012

The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus.

PubMed

Li, Jinlin; Callegari, Simone; Masucci, Maria G

2017-04-01

Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle.

The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus

PubMed Central

Li, Jinlin; Callegari, Simone

2017-01-01

Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle. PMID:28414785

RNA sequencing supports distinct reactive oxygen species-mediated pathways of apoptosis by high and low size mass fractions of Bay leaf (Lauris nobilis) in HT-29 cells.

PubMed

Rodd, Annabelle L; Ververis, Katherine; Sayakkarage, Dheeshana; Khan, Abdul W; Rafehi, Haloom; Ziemann, Mark; Loveridge, Shanon J; Lazarus, Ross; Kerr, Caroline; Lockett, Trevor; El-Osta, Assam; Karagiannis, Tom C; Bennett, Louise E

2015-08-01

Anti-proliferative and pro-apoptotic effects of Bay leaf (Laurus nobilis) in mammalian cancer and HT-29 adenocarcinoma cells have been previously attributed to effects of polyphenolic and essential oil chemical species. Recently, we demonstrated differentiated growth-regulating effects of high (HFBL) versus low molecular mass (LFBL) aqueous fractions of bay leaf and now confirm by comparative effects on gene expression, that HFBL and LFBL suppress HT-29 growth by distinct mechanisms. Induction of intra-cellular lesions including DNA strand breakage by extra-cellular HFBL, invoked the hypothesis that iron-mediated reactive oxygen species with capacity to penetrate cell membrane, were responsible for HFBL-mediated effects, supported by equivalent effects of HFBL in combination with γ radiation. Activities of HFBL and LFBL were interpreted to reflect differentiated responses to iron-mediated reactive oxygen species (ROS), occurring either outside or inside cells. In the presence of LFBL, apoptotic death was relatively delayed compared with HFBL. ROS production by LFBL mediated p53-dependent apoptosis and recovery was suppressed by promoting G1/S phase arrest and failure of cellular tight junctions. In comparison, intra-cellular anti-oxidant protection exerted by LFBL was absent for extra-cellular HFBL (likely polysaccharide-rich), which potentiated more rapid apoptosis by producing DNA double strand breaks. Differentiated effects on expression of genes regulating ROS defense and chromatic condensation by LFBL versus HFBL, were observed. The results support ferrous iron in cell culture systems and potentially in vivo, can invoke different extra-cellular versus intra-cellular ROS-mediated chemistries, that may be regulated by exogenous, including dietary species.

Structural basis for selectivity and diversity in angiotensin II receptors

DOE PAGES

Zhang, Haitao; Han, Gye Won; Batyuk, Alexander; ...

2017-04-20

The angiotensin II receptors AT 1R and AT 2R serve as key components of the renin–angiotensin–aldosterone system. AT 1R has a central role in the regulation of blood pressure, but the function of AT 2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT 2R bound to an AT 2R-selective ligand and to an AT 1R/AT 2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position,more » stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure–activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Finally, our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.« less

Structural basis for selectivity and diversity in angiotensin II receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Haitao; Han, Gye Won; Batyuk, Alexander

The angiotensin II receptors AT 1R and AT 2R serve as key components of the renin–angiotensin–aldosterone system. AT 1R has a central role in the regulation of blood pressure, but the function of AT 2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT 2R bound to an AT 2R-selective ligand and to an AT 1R/AT 2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position,more » stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure–activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Finally, our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.« less

Ubiquitin in Influenza Virus Entry and Innate Immunity.

PubMed

Rudnicka, Alina; Yamauchi, Yohei

2016-10-24

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle.

Ubiquitin in Influenza Virus Entry and Innate Immunity

PubMed Central

Rudnicka, Alina; Yamauchi, Yohei

2016-01-01

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle. PMID:27783058

Competence in Streptococcus pneumoniae is regulated by the rate of ribosomal decoding errors.

PubMed

Stevens, Kathleen E; Chang, Diana; Zwack, Erin E; Sebert, Michael E

2011-01-01

Competence for genetic transformation in Streptococcus pneumoniae develops in response to accumulation of a secreted peptide pheromone and was one of the initial examples of bacterial quorum sensing. Activation of this signaling system induces not only expression of the proteins required for transformation but also the production of cellular chaperones and proteases. We have shown here that activity of this pathway is sensitively responsive to changes in the accuracy of protein synthesis that are triggered by either mutations in ribosomal proteins or exposure to antibiotics. Increasing the error rate during ribosomal decoding promoted competence, while reducing the error rate below the baseline level repressed the development of both spontaneous and antibiotic-induced competence. This pattern of regulation was promoted by the bacterial HtrA serine protease. Analysis of strains with the htrA (S234A) catalytic site mutation showed that the proteolytic activity of HtrA selectively repressed competence when translational fidelity was high but not when accuracy was low. These findings redefine the pneumococcal competence pathway as a response to errors during protein synthesis. This response has the capacity to address the immediate challenge of misfolded proteins through production of chaperones and proteases and may also be able to address, through genetic exchange, upstream coding errors that cause intrinsic protein folding defects. The competence pathway may thereby represent a strategy for dealing with lesions that impair proper protein coding and for maintaining the coding integrity of the genome. The signaling pathway that governs competence in the human respiratory tract pathogen Streptococcus pneumoniae regulates both genetic transformation and the production of cellular chaperones and proteases. The current study shows that this pathway is sensitively controlled in response to changes in the accuracy of protein synthesis. Increasing the error rate during ribosomal decoding induced competence, while decreasing the error rate repressed competence. This pattern of regulation was promoted by the HtrA protease, which selectively repressed competence when translational fidelity was high but not when accuracy was low. Our findings demonstrate that this organism is able to monitor the accuracy of information used for protein biosynthesis and suggest that errors trigger a response addressing both the immediate challenge of misfolded proteins and, through genetic exchange, upstream coding errors that may underlie protein folding defects. This pathway may represent an evolutionary strategy for maintaining the coding integrity of the genome.

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes

PubMed Central

Newton, Matilda S.; Arcus, Vickery L.; Patrick, Wayne M.

2015-01-01

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks. PMID:25926697

Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging

PubMed Central

Pandey, Mukesh K.; DeGrado, Timothy R.

2016-01-01

Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed. PMID:26941849

Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner.

PubMed

Mitew, Stanislaw; Gobius, Ilan; Fenlon, Laura R; McDougall, Stuart J; Hawkes, David; Xing, Yao Lulu; Bujalka, Helena; Gundlach, Andrew L; Richards, Linda J; Kilpatrick, Trevor J; Merson, Tobias D; Emery, Ben

2018-01-22

Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.

Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.

2013-06-17

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significantmore » improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.« less

Robust Selection Algorithm (RSA) for Multi-Omic Biomarker Discovery; Integration with Functional Network Analysis to Identify miRNA Regulated Pathways in Multiple Cancers.

PubMed

Sehgal, Vasudha; Seviour, Elena G; Moss, Tyler J; Mills, Gordon B; Azencott, Robert; Ram, Prahlad T

2015-01-01

MicroRNAs (miRNAs) play a crucial role in the maintenance of cellular homeostasis by regulating the expression of their target genes. As such, the dysregulation of miRNA expression has been frequently linked to cancer. With rapidly accumulating molecular data linked to patient outcome, the need for identification of robust multi-omic molecular markers is critical in order to provide clinical impact. While previous bioinformatic tools have been developed to identify potential biomarkers in cancer, these methods do not allow for rapid classification of oncogenes versus tumor suppressors taking into account robust differential expression, cutoffs, p-values and non-normality of the data. Here, we propose a methodology, Robust Selection Algorithm (RSA) that addresses these important problems in big data omics analysis. The robustness of the survival analysis is ensured by identification of optimal cutoff values of omics expression, strengthened by p-value computed through intensive random resampling taking into account any non-normality in the data and integration into multi-omic functional networks. Here we have analyzed pan-cancer miRNA patient data to identify functional pathways involved in cancer progression that are associated with selected miRNA identified by RSA. Our approach demonstrates the way in which existing survival analysis techniques can be integrated with a functional network analysis framework to efficiently identify promising biomarkers and novel therapeutic candidates across diseases.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development.

PubMed

David, Aldana P; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B

2016-05-19

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development

PubMed Central

David, Aldana P.; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B.

2016-01-01

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology. PMID:26773060

Selective Amplification of the Genome Surrounding Key Placental Genes in Trophoblast Giant Cells.

PubMed

Hannibal, Roberta L; Baker, Julie C

2016-01-25

While most cells maintain a diploid state, polyploid cells exist in many organisms and are particularly prevalent within the mammalian placenta [1], where they can generate more than 900 copies of the genome [2]. Polyploidy is thought to be an efficient method of increasing the content of the genome by avoiding the costly and slow process of cytokinesis [1, 3, 4]. Polyploidy can also affect gene regulation by amplifying a subset of genomic regions required for specific cellular function [1, 3, 4]. This mechanism is found in the fruit fly Drosophila melanogaster, where polyploid ovarian follicle cells amplify genomic regions containing chorion genes, which facilitate secretion of eggshell proteins [5]. Here, we report that genomic amplification also occurs in mammals at selective regions of the genome in parietal trophoblast giant cells (p-TGCs) of the mouse placenta. Using whole-genome sequencing (WGS) and digital droplet PCR (ddPCR) of mouse p-TGCs, we identified five amplified regions, each containing a gene family known to be involved in mammalian placentation: the prolactins (two clusters), serpins, cathepsins, and the natural killer (NK)/C-type lectin (CLEC) complex [6-12]. We report here the first description of amplification at selective genomic regions in mammals and present evidence that this is an important mode of genome regulation in placental TGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aft2, a Novel Transcription Regulator, Is Required for Iron Metabolism, Oxidative Stress, Surface Adhesion and Hyphal Development in Candida albicans

PubMed Central

Xu, Ning; Cheng, Xinxin; Yu, Qilin; Qian, Kefan; Ding, Xiaohui; Liu, Ruming; Zhang, Biao; Xing, Laijun; Li, Mingchun

2013-01-01

Morphological transition and iron metabolism are closely relevant to Candida albicans pathogenicity and virulence. In our previous study, we demonstrated that C. albicans Aft2 plays an important role in ferric reductase activity and virulence. Here, we further explored the roles of C. albicans Aft2 in numerous cellular processes. We found that C. albicans Aft2 exhibited an important role in iron metabolism through bi-directional regulation effects on iron-regulon expression. Deletion of AFT2 reduced cellular iron accumulation under iron-deficient conditions. Furthermore, both reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity were remarkably increased in the aft2Δ/Δ mutant, which were thought to be responsible for the defective responses to oxidative stress. However, we found that over-expression of C. albicans AFT2 under the regulation of the strong PGK1 promoter could not effectively rescue Saccharomyces cerevisiae aft1Δ mutant defects in some cellular processes, such as cell-wall assembly, ion homeostasis and alkaline resistance, suggesting a possibility that C. albicans Aft2 weakened its functional role of regulating some cellular metabolism during the evolutionary process. Interestingly, deletion of AFT2 in C. albicans increased cell surface hydrophobicity, cell flocculation and the ability of adhesion to polystyrene surfaces. In addition, our results also revealed that C. albicans Aft2 played a dual role in regulating hypha-specific genes under solid and liquid hyphal inducing conditions. Deletion of AFT2 caused an impaired invasive growth in solid medium, but an increased filamentous aggregation and growth in liquid conditions. Moreover, iron deficiency and environmental cues induced nuclear import of Aft2, providing additional evidence for the roles of Aft2 in transcriptional regulation. PMID:23626810

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation

PubMed Central

Lawag, Abdalla A.; Napper, Jennifer M.; Hunter, Caroline A.; Bacon, Nickolas A.; Deskins, Seth; El-hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C.

2017-01-01

Abstract Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%–90% of the cells die when placed in medium where the major growth factor is granulocyte–macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer. PMID:28910138

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation.

PubMed

Lawag, Abdalla A; Napper, Jennifer M; Hunter, Caroline A; Bacon, Nickolas A; Deskins, Seth; El-Hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C; Sollars, Vincent E

2017-10-01

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.

Wise retained in the endoplasmic reticulum inhibits Wnt signaling by reducing cell surface LRP6.

PubMed

Guidato, Sonia; Itasaki, Nobue

2007-10-15

The Wnt signaling pathway is tightly regulated by extracellular and intracellular modulators. Wise was isolated as a secreted protein capable of interacting with the Wnt co-receptor LRP6. Studies in Xenopus embryos revealed that Wise either enhances or inhibits the Wnt pathway depending on the cellular context. Here we show that the cellular localization of Wise has distinct effects on the Wnt pathway readout. While secreted Wise either synergizes or inhibits the Wnt signals depending on the partner ligand, ER-retained Wise consistently blocks the Wnt pathway. ER-retained Wise reduces LRP6 on the cell surface, making cells less susceptible to the Wnt signal. This study provides a cellular mechanism for the action of Wise and introduces the modulation of cellular susceptibility to Wnt signals as a novel mechanism of the regulation of the Wnt pathway.

Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence*

PubMed Central

Tsujii, Akira; Miyamoto, Yoichi; Moriyama, Tetsuji; Tsuchiya, Yuko; Obuse, Chikashi; Mizuguchi, Kenji; Oka, Masahiro; Yoneda, Yoshihiro

2015-01-01

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence. PMID:26491019

Control of fluxes in metabolic networks

PubMed Central

Basler, Georg; Nikoloski, Zoran; Larhlimi, Abdelhalim; Barabási, Albert-László; Liu, Yang-Yu

2016-01-01

Understanding the control of large-scale metabolic networks is central to biology and medicine. However, existing approaches either require specifying a cellular objective or can only be used for small networks. We introduce new coupling types describing the relations between reaction activities, and develop an efficient computational framework, which does not require any cellular objective for systematic studies of large-scale metabolism. We identify the driver reactions facilitating control of 23 metabolic networks from all kingdoms of life. We find that unicellular organisms require a smaller degree of control than multicellular organisms. Driver reactions are under complex cellular regulation in Escherichia coli, indicating their preeminent role in facilitating cellular control. In human cancer cells, driver reactions play pivotal roles in malignancy and represent potential therapeutic targets. The developed framework helps us gain insights into regulatory principles of diseases and facilitates design of engineering strategies at the interface of gene regulation, signaling, and metabolism. PMID:27197218

Activation of cellular death programs associated with immunosenescence-like phenotype in TPPII knockout mice

PubMed Central

Huai, Jisen; Firat, Elke; Nil, Ahmed; Million, Daniele; Gaedicke, Simone; Kanzler, Benoit; Freudenberg, Marina; van Endert, Peter; Kohler, Gabriele; Pahl, Heike L.; Aichele, Peter; Eichmann, Klaus; Niedermann, Gabriele

2008-01-01

The giant cytosolic protease tripeptidyl peptidase II (TPPII) has been implicated in the regulation of proliferation and survival of malignant cells, particularly lymphoma cells. To address its functions in normal cellular and systemic physiology we have generated TPPII-deficient mice. TPPII deficiency activates cell type-specific death programs, including proliferative apoptosis in several T lineage subsets and premature cellular senescence in fibroblasts and CD8+ T cells. This coincides with up-regulation of p53 and dysregulation of NF-κB. Prominent degenerative alterations at the organismic level were a decreased lifespan and symptoms characteristic of immunohematopoietic senescence. These symptoms include accelerated thymic involution, lymphopenia, impaired proliferative T cell responses, extramedullary hematopoiesis, and inflammation. Thus, TPPII is important for maintaining normal cellular and systemic physiology, which may be relevant for potential therapeutic applications of TPPII inhibitors. PMID:18362329

Dusp5 negatively regulates IL-33-mediated eosinophil survival and function

PubMed Central

Holmes, Derek A; Yeh, Jung-Hua; Yan, Donghong; Xu, Min; Chan, Andrew C

2015-01-01

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5−/− mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5−/− eosinophils exhibit increased cellular ERK1/2 activation and BCL-XL expression that results in enhanced eosinophil survival. In addition, Dusp5−/− eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival. PMID:25398911

Redox signaling: Potential arbitrator of autophagy and apoptosis in therapeutic response.

PubMed

Zhang, Lu; Wang, Kui; Lei, Yunlong; Li, Qifu; Nice, Edouard Collins; Huang, Canhua

2015-12-01

Redox signaling plays important roles in the regulation of cell death and survival in response to cancer therapy. Autophagy and apoptosis are discrete cellular processes mediated by distinct groups of regulatory and executioner molecules, and both are thought to be cellular responses to various stress conditions including oxidative stress, therefore controlling cell fate. Basic levels of reactive oxygen species (ROS) may function as signals to promote cell proliferation and survival, whereas increase of ROS can induce autophagy and apoptosis by damaging cellular components. Growing evidence in recent years argues for ROS that below detrimental levels acting as intracellular signal transducers that regulate autophagy and apoptosis. ROS-regulated autophagy and apoptosis can cross-talk with each other. However, how redox signaling determines different cell fates by regulating autophagy and apoptosis remains unclear. In this review, we will focus on understanding the delicate molecular mechanism by which autophagy and apoptosis are finely orchestrated by redox signaling and discuss how this understanding can be used to develop strategies for the treatment of cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

ROS-mediated redox signaling during cell differentiation in plants.

PubMed

Schmidt, Romy; Schippers, Jos H M

2015-08-01

Reactive oxygen species (ROS) have emerged in recent years as important regulators of cell division and differentiation. The cellular redox state has a major impact on cell fate and multicellular organism development. However, the exact molecular mechanisms through which ROS manifest their regulation over cellular development are only starting to be understood in plants. ROS levels are constantly monitored and any change in the redox pool is rapidly sensed and responded upon. Different types of ROS cause specific oxidative modifications, providing the basic characteristics of a signaling molecule. Here we provide an overview of ROS sensors and signaling cascades that regulate transcriptional responses in plants to guide cellular differentiation and organ development. Although several redox sensors and cascades have been identified, they represent only a first glimpse on the impact that redox signaling has on plant development and growth. We provide an initial evaluation of ROS signaling cascades involved in cell differentiation in plants and identify potential avenues for future studies. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Ral GTPase and the exocyst regulate autophagy in a tissue-specific manner.

PubMed

Tracy, Kirsten; Velentzas, Panagiotis D; Baehrecke, Eric H

2016-01-01

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress-induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation-induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context-dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch. © 2015 The Authors.

Steap4 Plays a Critical Role in Osteoclastogenesis in Vitro by Regulating Cellular Iron/Reactive Oxygen Species (ROS) Levels and cAMP Response Element-binding Protein (CREB) Activation*

PubMed Central

Zhou, Jian; Ye, Shiqiao; Fujiwara, Toshifumi; Manolagas, Stavros C.; Zhao, Haibo

2013-01-01

Iron is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with excessive bone resorption. Iron uptake by osteoclast precursors via the transferrin cycle increases mitochondrial biogenesis, reactive oxygen species production, and activation of cAMP response element-binding protein, a critical transcription factor downstream of receptor activator of NF-κB-ligand-induced calcium signaling. These changes are required for the differentiation of osteoclast precursors to mature bone-resorbing osteoclasts. However, the molecular mechanisms regulating cellular iron metabolism in osteoclasts remain largely unknown. In this report, we provide evidence that Steap4, a member of the six-transmembrane epithelial antigen of prostate (Steap) family proteins, is an endosomal ferrireductase with a critical role in cellular iron utilization in osteoclasts. Specifically, we show that Steap4 is the only Steap family protein that is up-regulated during osteoclast differentiation. Knocking down Steap4 expression in vitro by lentivirus-mediated short hairpin RNAs inhibits osteoclast formation and decreases cellular ferrous iron, reactive oxygen species, and the activation of cAMP response element-binding protein. These results demonstrate that Steap4 is a critical enzyme for cellular iron uptake and utilization in osteoclasts and, thus, indispensable for osteoclast development and function. PMID:23990467

Regulation of BolA abundance mediates morphogenesis in Fremyella diplosiphon

DOE PAGES

Singh, Shailendra P.; Montgomery, Beronda L.

2015-11-05

Filamentous cyanobacterium Fremyella diplosiphon is known to alter its pigmentation and morphology during complementary chromatic acclimation (CCA) to efficiently harvest available radiant energy for photosynthesis. F. diplosiphon cells are rectangular and filaments are longer under green light (GL), whereas smaller, spherical cells and short filaments are prevalent under red light (RL). Light regulation of bolA morphogene expression is correlated with photoregulation of cellular morphology in F. diplosiphon. Here, we investigate a role for quantitative regulation of cellular BolA protein levels in morphology determination. Overexpression of bolA in WT was associated with induction of RL-characteristic spherical morphology even when cultures weremore » grown under GL. Overexpression of bolA in a ΔrcaE background, which lacks cyanobacteriochrome photosensor RcaE and accumulates lower levels of BolA than WT, partially reverted the cellular morphology of the strain to a WT-like state. Overexpression of BolA in WT and ΔrcaE backgrounds was associated with decreased cellular reactive oxygen species (ROS) levels and an increase in filament length under both GL and RL. Morphological defects and high ROS levels commonly observed in ΔrcaE could, thus, be in part due to low accumulation of BolA. Together, these findings support an emerging model for RcaE-dependent photoregulation of BolA in controlling the cellular morphology of F. diplosiphon during CCA.« less

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA*

PubMed Central

Wigington, Callie P.; Morris, Kevin J.; Newman, Laura E.; Corbett, Anita H.

2016-01-01

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function. PMID:27563065

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus.

PubMed

Biggar, Kyle K; Wu, Cheng-Wei; Tessier, Shannon N; Zhang, Jing; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B

2015-04-01

Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK) family members to primate torpor were compared in six organs of control (aroused) versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs), c-jun NH2-terminal kinases (JNKs), MAPK kinase (MEK), and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27). The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor. Copyright © 2015. Production and hosting by Elsevier Ltd.

Distinct functional outputs of PTEN signalling are controlled by dynamic association with β-arrestins

PubMed Central

Lima-Fernandes, Evelyne; Enslen, Hervé; Camand, Emeline; Kotelevets, Larissa; Boularan, Cédric; Achour, Lamia; Benmerah, Alexandre; Gibson, Lucien C D; Baillie, George S; Pitcher, Julie A; Chastre, Eric; Etienne-Manneville, Sandrine; Marullo, Stefano; Scott, Mark G H

2011-01-01

The tumour suppressor PTEN (phosphatase and tensin deleted on chromosome 10) regulates major cellular functions via lipid phosphatase-dependent and -independent mechanisms. Despite its fundamental pathophysiological importance, how PTEN's cellular activity is regulated has only been partially elucidated. We report that the scaffolding proteins β-arrestins (β-arrs) are important regulators of PTEN. Downstream of receptor-activated RhoA/ROCK signalling, β-arrs activate the lipid phosphatase activity of PTEN to negatively regulate Akt and cell proliferation. In contrast, following wound-induced RhoA activation, β-arrs inhibit the lipid phosphatase-independent anti-migratory effects of PTEN. β-arrs can thus differentially control distinct functional outputs of PTEN important for cell proliferation and migration. PMID:21642958

Selective Vasopressin-1a receptor antagonist prevents brain edema, reduces astrocytic cell swelling and GFAP, V1aR and AQP4 expression after focal traumatic brain injury

PubMed Central

Marmarou, Christina R.; Liang, Xiuyin; Abidi, Naqeeb H.; Parveen, Shanaz; Taya, Keisuke; Henderson, Scott C.; Young, Harold F.; Filippidis, Aristotelis S.; Baumgarten, Clive M.

2014-01-01

A secondary and often lethal consequence of traumatic brain injury is cellular edema that we posit is due to astrocytic swelling caused by transmembrane water fluxes augmented by vasopressin-regulated aquaporin-4 (AQP4). We therefore tested whether vasopressin 1a receptor (V1aR) inhibition would suppress astrocyte AQP4, reduce astrocytic edema, and thereby diminish TBI-induced edematous changes. V1aR inhibition by SR49059 significantly reduced brain edema after cortical contusion injury (CCI) in rat 5 h post-injury. Injured-hemisphere brain water content (n=6 animals/group) and astrocytic area (n=3/group) were significantly higher in CCI-vehicle (80.5±0.3%; 18.0±1.4 µm2) versus sham groups (78.3±0.1%; 9.5±0.9 µm2), and SR49059 blunted CCI-induced increases in brain edema (79.0±0.2%; 9.4±0.8 µm2). CCI significantly up-regulated GFAP, V1aR and AQP4 protein levels and SR49059 suppressed injury induced up regulation (n=6/group). In CCI-vehicle, sham and CCI-SR49059 groups, GFAP was 1.58±0.04, 0.47±0.02, and 0.81±0.03, respectively; V1aR was 1.00±0.06, 0.45±0.05, and 0.46±0.09; and AQP4 was 2.03± 0.34, 0.49±0.04, and 0.92±0.22. Confocal immunohistochemistry gave analogous results. In CCI-vehicle, sham and CCI-SR49059 groups, fluorescence intensity of GFAP was 349±38, 56±5, and 244±30, respectively, V1aR was 601±71, 117.8±14, and 390±76, and AQP4 was 818±117, 158±5, and 458±55 (n=3/group). The results support that edema was predominantly cellular following CCI and documented that V1aR inhibition with SR49059 suppressed injury-induced up regulation of GFAP, V1A and AQP4, blunting edematous changes. Our findings suggest V1aR inhibitors may be potential therapeutic tools to prevent cellular swelling and provide treatment for post-traumatic brain edema. PMID:24933327

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

PubMed

Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

2017-09-01

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Surviving the cold: molecular analyses of insect cryoprotective dehydration in the Arctic springtail Megaphorura arctica (Tullberg)

PubMed Central

Clark, Melody S; Thorne, Michael AS; Purać, Jelena; Burns, Gavin; Hillyard, Guy; Popović, Željko D; Grubor-Lajšić, Gordana; Worland, M Roger

2009-01-01

Background Insects provide tractable models for enhancing our understanding of the physiological and cellular processes that enable survival at extreme low temperatures. They possess three main strategies to survive the cold: freeze tolerance, freeze avoidance or cryoprotective dehydration, of which the latter method is exploited by our model species, the Arctic springtail Megaphorura arctica, formerly Onychiurus arcticus (Tullberg 1876). The physiological mechanisms underlying cryoprotective dehydration have been well characterised in M. arctica and to date this process has been described in only a few other species: the Antarctic nematode Panagrolaimus davidi, an enchytraied worm, the larvae of the Antarctic midge Belgica antarctica and the cocoons of the earthworm Dendrobaena octaedra. There are no in-depth molecular studies on the underlying cold survival mechanisms in any species. Results A cDNA microarray was generated using 6,912 M. arctica clones printed in duplicate. Analysis of clones up-regulated during dehydration procedures (using both cold- and salt-induced dehydration) has identified a number of significant cellular processes, namely the production and mobilisation of trehalose, protection of cellular systems via small heat shock proteins and tissue/cellular remodelling during the dehydration process. Energy production, initiation of protein translation and cell division, plus potential tissue repair processes dominate genes identified during recovery. Heat map analysis identified a duplication of the trehalose-6-phosphate synthase (TPS) gene in M. arctica and also 53 clones co-regulated with TPS, including a number of membrane associated and cell signalling proteins. Q-PCR on selected candidate genes has also contributed to our understanding with glutathione-S-transferase identified as the major antioxdidant enzyme protecting the cells during these stressful procedures, and a number of protein kinase signalling molecules involved in recovery. Conclusion Microarray analysis has proved to be a powerful technique for understanding the processes and genes involved in cryoprotective dehydration, beyond the few candidate genes identified in the current literature. Dehydration is associated with the mobilisation of trehalose, cell protection and tissue remodelling. Energy production, leading to protein production, and cell division characterise the recovery process. Novel membrane proteins, along with aquaporins and desaturases, have been identified as promising candidates for future functional analyses to better understand membrane remodelling during cellular dehydration. PMID:19622137

Regulation of cellular growth by the Drosophila target of rapamycin dTOR

PubMed Central

Zhang, Hongbing; Stallock, James P.; Ng, Joyce C.; Reinhard, Christoph; Neufeld, Thomas P.

2000-01-01

The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. Here, we show that the Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of p70 S6 kinase (p70S6K) in vitro, and we demonstrate that overexpression of p70S6K in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. Our results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability. PMID:11069888

Antagonistic regulation, yet synergistic defense: effect of bergapten and protease inhibitor on development of cowpea bruchid Callosobruchus maculatus.

PubMed

Guo, Fengguang; Lei, Jiaxin; Sun, Yucheng; Chi, Yong Hun; Ge, Feng; Patil, Bhimanagouda S; Koiwa, Hisashi; Zeng, Rensen; Zhu-Salzman, Keyan

2012-01-01

The furanocoumarin compound bergapten is a plant secondary metabolite that has anti-insect function. When incorporated into artificial diet, it retarded cowpea bruchid development, decreased fecundity, and caused mortality at a sufficient dose. cDNA microarray analysis indicated that cowpea bruchid altered expression of 543 midgut genes in response to dietary bergapten. Among these bergapten-regulated genes, 225 have known functions; for instance, those encoding proteins related to nutrient transport and metabolism, development, detoxification, defense and various cellular functions. Such differential gene regulation presumably facilitates the bruchids' countering the negative effect of dietary bergapten. Many genes did not have homology (E-value cutoff 10(-6)) with known genes in a BlastX search (206), or had homology only with genes of unknown function (112). Interestingly, when compared with the transcriptomic profile of cowpea bruchids treated with dietary soybean cysteine protease inhibitor N (scN), 195 out of 200 coregulated midgut genes are oppositely regulated by the two compounds. Simultaneous administration of bergapten and scN attenuated magnitude of change in selected oppositely-regulated genes, as well as led to synergistic delay in insect development. Therefore, targeting insect vulnerable sites that may compromise each other's counter-defensive response has the potential to increase the efficacy of the anti-insect molecules.

Role of Mitochondrial Ca2+ in the Regulation of Cellular Energetics

PubMed Central

Glancy, Brian; Balaban, Robert S.

2012-01-01

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca2+ ion membrane gradients make Ca2+ signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca2+ regulates many cellular ATP consuming reactions such as muscle contraction, exocytosis, biosynthesis and neuronal signaling. Thus, Ca2+ becomes a logical candidate as a signaling molecule to modulate ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient across their inner membrane providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial [Ca2+], identification of transport mechanisms as well as proximity of mitochondria to Ca2+ release sites further supports the notion that Ca2+ can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca2+ plays a role in the regulation of ATP generation and potentially contributes to the orchestration of the cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca2+, which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca2+ on mitochondrial energy conversion. Numerous non-invasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption and workloads suggest significant Ca2+ effects on other elements of NADH generation as well as downstream elements of oxidative phosphorylation including the F1FO-ATPase and the cytochrome chain. These other potential elements of Ca2+ modification of mitochondrial energy conversion will be the focus of this review. Though most of specific molecular mechanisms have yet to be elucidated, it is clear that Ca2+ provides a balanced activation of mitochondrial energy metabolism which exceeds the alteration of dehydrogenases alone. PMID:22443365

The Role of microRNAs in the Pathogenesis of Herpesvirus Infection.

PubMed

Piedade, Diogo; Azevedo-Pereira, José Miguel

2016-06-02

MicroRNAs (miRNAs) are small non-coding RNAs important in gene regulation. They are able to regulate mRNA translation through base-pair complementarity. Cellular miRNAs have been involved in the regulation of nearly all cellular pathways, and their deregulation has been associated with several diseases such as cancer. Given the importance of microRNAs to cell homeostasis, it is no surprise that viruses have evolved to take advantage of this cellular pathway. Viruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. Moreover, viral inhibition of key proteins from the microRNA pathway and important changes in cellular microRNA pool have been reported upon viral infection. In addition, viruses have developed multiple mechanisms to avoid being targeted by cellular microRNAs. This complex interaction between host and viruses to control the microRNA pathway usually favors viral infection and persistence by either reducing immune detection, avoiding apoptosis, promoting cell growth, or promoting lytic or latent infection. One of the best examples of this virus-host-microRNA interplay emanates from members of the Herperviridae family, namely the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), human herpesvirus 8 (HHV-8), and the Epstein-Barr virus (EBV). In this review, we will focus on the general functions of microRNAs and the interactions between herpesviruses, human hosts, and microRNAs and will delve into the related mechanisms that contribute to infection and pathogenesis.

The Role of microRNAs in the Pathogenesis of Herpesvirus Infection

PubMed Central

Piedade, Diogo; Azevedo-Pereira, José Miguel

2016-01-01

MicroRNAs (miRNAs) are small non-coding RNAs important in gene regulation. They are able to regulate mRNA translation through base-pair complementarity. Cellular miRNAs have been involved in the regulation of nearly all cellular pathways, and their deregulation has been associated with several diseases such as cancer. Given the importance of microRNAs to cell homeostasis, it is no surprise that viruses have evolved to take advantage of this cellular pathway. Viruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. Moreover, viral inhibition of key proteins from the microRNA pathway and important changes in cellular microRNA pool have been reported upon viral infection. In addition, viruses have developed multiple mechanisms to avoid being targeted by cellular microRNAs. This complex interaction between host and viruses to control the microRNA pathway usually favors viral infection and persistence by either reducing immune detection, avoiding apoptosis, promoting cell growth, or promoting lytic or latent infection. One of the best examples of this virus-host-microRNA interplay emanates from members of the Herperviridae family, namely the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), human herpesvirus 8 (HHV-8), and the Epstein–Barr virus (EBV). In this review, we will focus on the general functions of microRNAs and the interactions between herpesviruses, human hosts, and microRNAs and will delve into the related mechanisms that contribute to infection and pathogenesis. PMID:27271654

Interface Pattern Selection Criterion for Cellular Structures in Directional Solidification

NASA Technical Reports Server (NTRS)

Trivedi, R.; Tewari, S. N.; Kurtze, D.

1999-01-01

The aim of this investigation is to establish key scientific concepts that govern the selection of cellular and dendritic patterns during the directional solidification of alloys. We shall first address scientific concepts that are crucial in the selection of interface patterns. Next, the results of ground-based experimental studies in the Al-4.0 wt % Cu system will be described. Both experimental studies and theoretical calculations will be presented to establish the need for microgravity experiments.

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, J.; Pei-Chen Lin, C.; Pathak, M. C.

2016-07-06

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumedmore » during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.« less

Sequential Axon-derived Signals Couple Target Survival and Layer Specificity in the Drosophila Visual System

PubMed Central

Pecot, Matthew Y.; Chen, Yi; Akin, Orkun; Chen, Zhenqing; Tsui, C.Y. Kimberly; Zipursky, S. Lawrence

2015-01-01

SUMMARY Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1–R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. Here we demonstrate that additional axon-derived signals selectively couple target survival with layer-specificity. We show that Jelly belly (Jeb) produced by R1–R6 axons interacts with its receptor, anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. We propose that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system. PMID:24742459

THE HISTONE DEACETYLASE SIRT6 IS A NOVEL TUMOR SUPPRESSOR THAT CONTROLS CANCER METABOLISM

PubMed Central

Sebastián, Carlos; Zwaans, Bernardette M. M.; Silberman, Dafne M.; Gymrek, Melissa; Goren, Alon; Zhong, Lei; Ram, Oren; Truelove, Jessica; Guimaraes, Alexander R.; Toiber, Debra; Cosentino, Claudia; Greenson, Joel K.; Mac Donald, Alasdair I; McGlynn, Liane; Maxwell, Fraser; Edwards, Joanne; Giacosa, Sofia; Guccione, Ernesto; Weissleder, Ralph; Bernstein, Bradley E.; Regev, Aviv; Shiels, Paul G.; Lombard, David B.; Mostoslavsky, Raul

2012-01-01

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, while transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. Using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size and aggressiveness of tumors. SIRT6 also functions as a novel regulator of ribosome metabolism by co-repressing MYC transcriptional activity. Lastly, SIRT6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism. PMID:23217706

Understanding α-globin gene regulation and implications for the treatment of β-thalassemia.

PubMed

Mettananda, Sachith; Gibbons, Richard J; Higgs, Douglas R

2016-03-01

Over the past three decades, a vast amount of new information has been uncovered describing how the globin genes are regulated. This knowledge has provided significant insights into the general understanding of the regulation of human genes. It is now known that molecular defects within and around the α- and β-globin genes, as well as in the distant regulatory elements, can cause thalassemia. Unbalanced production of globin chains owing to defective synthesis of one, and the continued unopposed synthesis of another, is the central causative factor in the cellular pathology and pathophysiology of thalassemia. A large body of clinical, genetic, and experimental evidence suggests that altering globin chain imbalance by reducing the production of α-globin synthesis ameliorates the disease severity in patients with β-thalassemia. With the development of new genetic-based therapeutic tools that have a potential to decrease the expression of a selected gene in a tissue-specific manner, the possibility of decreasing expression of the α-globin gene to improve the clinical severity of β-thalassemia could become a reality. © 2015 New York Academy of Sciences.

Selective reversible inhibition of autophagy in hypoxic breast cancer cells promotes pulmonary metastasis

PubMed Central

Dower, Christopher M.; Bhat, Neema; Wang, Edward W.; Wang, Hong-Gang

2016-01-01

Autophagy influences how cancer cells respond to nutrient deprivation and hypoxic stress, two hallmarks of the tumor microenvironment (TME). In this study, we explored the impact of autophagy on the pathophysiology of breast cancer cells, using a novel hypoxia-dependent, reversible dominant negative strategy to regulate autophagy at the cellular level within the TME. Suppression of autophagy via hypoxia-induced expression of the kinase-dead unc-51 like autophagy activating kinase (ULK1) mutant K46N increased lung metastases in MDA-MB-231 xenograft mouse models. Consistent with this effect, expressing a dominant-negative mutant of ULK1 or ATG4b or a ULK1-targeting shRNA facilitated cell migration in vitro. Functional proteomic and transcriptome analysis revealed that loss of hypoxia-regulated autophagy promotes metastasis via induction of the fibronectin integrin signaling axis. Indeed, loss of ULK1 function increased fibronectin deposition in the hypoxic TME. Together, our results indicated that hypoxia-regulated autophagy suppresses metastasis in breast cancer by preventing tumor fibrosis. These results also suggest cautions in the development of autophagy-based strategies for cancer treatment. PMID:28115361

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Jihui; Lin, Coney Pei-Chen; Pathak, Manish C.

2014-07-11

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumedmore » during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.« less

REVIEW ARTICLE: How do biomolecular systems speed up and regulate rates?

NASA Astrophysics Data System (ADS)

Zhou, Huan-Xiang

2005-09-01

The viability of a biological system depends upon careful regulation of the rates of various processes. These rates have limits imposed by intrinsic chemical or physical steps (e.g., diffusion). These limits can be expanded by interactions and dynamics of the biomolecules. For example, (a) a chemical reaction is catalyzed when its transition state is preferentially bound to an enzyme; (b) the folding of a protein molecule is speeded up by specific interactions within the transition-state ensemble and may be assisted by molecular chaperones; (c) the rate of specific binding of a protein molecule to a cellular target can be enhanced by mechanisms such as long-range electrostatic interactions, nonspecific binding and folding upon binding; (d) directional movement of motor proteins is generated by capturing favorable Brownian motion through intermolecular binding energy; and (e) conduction and selectivity of ions through membrane channels are controlled by interactions and the dynamics of channel proteins. Simple physical models are presented here to illustrate these processes and provide a unifying framework for understanding speed attainment and regulation in biomolecular systems.

Lhx2 Expression in Postmitotic Cortical Neurons Initiates Assembly of the Thalamocortical Somatosensory Circuit.

PubMed

Wang, Chia-Fang; Hsing, Hsiang-Wei; Zhuang, Zi-Hui; Wen, Meng-Hsuan; Chang, Wei-Jen; Briz, Carlos G; Nieto, Marta; Shyu, Bai Chuang; Chou, Shen-Ju

2017-01-24

Cortical neurons must be specified and make the correct connections during development. Here, we examine a mechanism initiating neuronal circuit formation in the barrel cortex, a circuit comprising thalamocortical axons (TCAs) and layer 4 (L4) neurons. When Lhx2 is selectively deleted in postmitotic cortical neurons using conditional knockout (cKO) mice, L4 neurons in the barrel cortex are initially specified but fail to form cellular barrels or develop polarized dendrites. In Lhx2 cKO mice, TCAs from the thalamic ventral posterior nucleus reach the barrel cortex but fail to further arborize to form barrels. Several activity-regulated genes and genes involved in regulating barrel formation are downregulated in the Lhx2 cKO somatosensory cortex. Among them, Btbd3, an activity-regulated gene controlling dendritic development, is a direct downstream target of Lhx2. We find that Lhx2 confers neuronal competency for activity-dependent dendritic development in L4 neurons by inducing the expression of Btbd3. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Complement Evasion Strategies of Viruses: An Overview

PubMed Central

Agrawal, Palak; Nawadkar, Renuka; Ojha, Hina; Kumar, Jitendra; Sahu, Arvind

2017-01-01

Being a major first line of immune defense, the complement system keeps a constant vigil against viruses. Its ability to recognize large panoply of viruses and virus-infected cells, and trigger the effector pathways, results in neutralization of viruses and killing of the infected cells. This selection pressure exerted by complement on viruses has made them evolve a multitude of countermeasures. These include targeting the recognition molecules for the avoidance of detection, targeting key enzymes and complexes of the complement pathways like C3 convertases and C5b-9 formation – either by encoding complement regulators or by recruiting membrane-bound and soluble host complement regulators, cleaving complement proteins by encoding protease, and inhibiting the synthesis of complement proteins. Additionally, viruses also exploit the complement system for their own benefit. For example, they use complement receptors as well as membrane regulators for cellular entry as well as their spread. Here, we provide an overview on the complement subversion mechanisms adopted by the members of various viral families including Poxviridae, Herpesviridae, Adenoviridae, Flaviviridae, Retroviridae, Picornaviridae, Astroviridae, Togaviridae, Orthomyxoviridae and Paramyxoviridae. PMID:28670306

Use of whole genome expression analysis in the toxicity screening of nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fröhlich, Eleonore, E-mail: eleonore.froehlich@medunigraz.at; Meindl, Claudia; Wagner, Karin

2014-10-15

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays formore » NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay.« less

Riboregulation of the bacterial actin-homolog MreB by DsrA small noncoding RNA.

PubMed

Cayrol, Bastien; Fortas, Emilie; Martret, Claire; Cech, Grzegorz; Kloska, Anna; Caulet, Stephane; Barbet, Marion; Trépout, Sylvain; Marco, Sergio; Taghbalout, Aziz; Busi, Florent; Wegrzyn, Grzegorz; Arluison, Véronique

2015-01-01

The bacterial actin-homolog MreB is a key player in bacterial cell-wall biosynthesis and is required for the maintenance of the rod-like morphology of Escherichia coli. However, how MreB cellular levels are adjusted to growth conditions is poorly understood. Here, we show that DsrA, an E. coli small noncoding RNA (sRNA), is involved in the post-transcriptional regulation of mreB. DsrA is required for the downregulation of MreB cellular concentration during environmentally induced slow growth-rates, mainly growth at low temperature and during the stationary phase. DsrA interacts in an Hfq-dependent manner with the 5' region of mreB mRNA, which contains signals for translation initiation and thereby affects mreB translation and stability. Moreover, as DsrA is also involved in the regulation of two transcriptional regulators, σ(S) and the nucleoid associated protein H-NS, which negatively regulate mreB transcription, it also indirectly contributes to mreB transcriptional down-regulation. By using quantitative analyses, our results evidence the complexity of this regulation and the tangled interplay between transcriptional and post-transcriptional control. As transcription factors and sRNA-mediated post-transcriptional regulators use different timescales, we propose that the sRNA pathway helps to adapt to changes in temperature, but also indirectly mediates long-term regulation of MreB concentration. The tight regulation and fine-tuning of mreB gene expression in response to cellular stresses is discussed in regard to the effect of the MreB protein on cell elongation.

Differential regulation of Smad3 and of the type II transforming growth factor-β receptor in mitosis: implications for signaling.

PubMed

Hirschhorn, Tal; Barizilay, Lior; Smorodinsky, Nechama I; Ehrlich, Marcelo

2012-01-01

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis.

Differential Regulation of Smad3 and of the Type II Transforming Growth Factor-β Receptor in Mitosis: Implications for Signaling

PubMed Central

Hirschhorn, Tal; Barizilay, Lior; Smorodinsky, Nechama I.; Ehrlich, Marcelo

2012-01-01

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis. PMID:22927969

Interferon Regulatory Factors IRF5 and IRF7 Inhibit Growth and Induce Senescence in Immortal Li-Fraumeni Fibroblasts

PubMed Central

Li, Qunfang; Tang, Lin; Roberts, Paul Christopher; Kraniak, Janice M.; Fridman, Aviva Levine; Kulaeva, Olga I.; Tehrani, Omid S.; Tainsky, Michael A.

2013-01-01

Cellular immortalization is one of the prerequisite steps in carcinogenesis. By gene expression profiling, we have found that genes in the interferon (IFN) pathway were dysregulated during the spontaneous cellular immortalization of fibroblasts from Li-Fraumeni syndrome (LFS) patients with germ-line mutations in p53. IFN signaling pathway genes were down-regulated by epigenetic silencing during immortalization, and some of these same IFN-regulated genes were activated during replicative senescence. Bisulfite sequencing of the promoter regions of two IFN regulatory transcription factors (IRF5 and IRF7) revealed that IRF7, but not IRF5, was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The induction of IRF7 gene by IFNα in immortal LFS cells was potentiated by pretreatment with the demethylation agent 5-aza-2′-deoxycytidine. Overexpression of IRF5 and IRF7 revealed that they can act either alone or in tandem to activate other IFN-regulated genes. In addition, they serve to inhibit the proliferation rate and induce a senescence-related phenotype in immortal LFS cells. Furthermore, polyinosinic:polycytidylic acid treatment of the IRF-overexpressing cells showed a more rapid induction of several IFN-regulated genes. We conclude that the epigenetic inactivation of the IFN pathway plays a critical role in cellular immortalization, and the reactivation of IFN-regulated genes by transcription factors IRF5 and/or IRF7 is sufficient to induce cellular senescence. The IFN pathway may provide valuable molecular targets for therapeutic interventions at early stages of cancer development. PMID:18505922

Interferon regulatory factors IRF5 and IRF7 inhibit growth and induce senescence in immortal Li-Fraumeni fibroblasts.

PubMed

Li, Qunfang; Tang, Lin; Roberts, Paul Christopher; Kraniak, Janice M; Fridman, Aviva Levine; Kulaeva, Olga I; Tehrani, Omid S; Tainsky, Michael A

2008-05-01

Cellular immortalization is one of the prerequisite steps in carcinogenesis. By gene expression profiling, we have found that genes in the interferon (IFN) pathway were dysregulated during the spontaneous cellular immortalization of fibroblasts from Li-Fraumeni syndrome (LFS) patients with germ-line mutations in p53. IFN signaling pathway genes were down-regulated by epigenetic silencing during immortalization, and some of these same IFN-regulated genes were activated during replicative senescence. Bisulfite sequencing of the promoter regions of two IFN regulatory transcription factors (IRF5 and IRF7) revealed that IRF7, but not IRF5, was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The induction of IRF7 gene by IFNalpha in immortal LFS cells was potentiated by pretreatment with the demethylation agent 5-aza-2'-deoxycytidine. Overexpression of IRF5 and IRF7 revealed that they can act either alone or in tandem to activate other IFN-regulated genes. In addition, they serve to inhibit the proliferation rate and induce a senescence-related phenotype in immortal LFS cells. Furthermore, polyinosinic:polycytidylic acid treatment of the IRF-overexpressing cells showed a more rapid induction of several IFN-regulated genes. We conclude that the epigenetic inactivation of the IFN pathway plays a critical role in cellular immortalization, and the reactivation of IFN-regulated genes by transcription factors IRF5 and/or IRF7 is sufficient to induce cellular senescence. The IFN pathway may provide valuable molecular targets for therapeutic interventions at early stages of cancer development.

Cellular and molecular basis for stress-induced depression.

PubMed

Seo, J-S; Wei, J; Qin, L; Kim, Y; Yan, Z; Greengard, P

2017-10-01

Chronic stress has a crucial role in the development of psychiatric diseases, such as anxiety and depression. Dysfunction of the medial prefrontal cortex (mPFC) has been linked to the cognitive and emotional deficits induced by stress. However, little is known about the molecular and cellular determinants in mPFC for stress-associated mental disorders. Here we show that chronic restraint stress induces the selective loss of p11 (also known as annexin II light chain, S100A10), a multifunctional protein binding to 5-HT receptors, in layer II/III neurons of the prelimbic cortex (PrL), as well as depression-like behaviors, both of which are reversed by selective serotonin reuptake inhibitors (SSRIs) and the tricyclic class of antidepressant (TCA) agents. In layer II/III of the PrL, p11 is highly concentrated in dopamine D2 receptor-expressing (D2 + ) glutamatergic neurons. Viral expression of p11 in D2 + PrL neurons alleviates the depression-like behaviors exhibited by genetically manipulated mice with D2 + neuron-specific or global deletion of p11. In stressed animals, overexpression of p11 in D2 + PrL neurons rescues depression-like behaviors by restoring glutamatergic transmission. Our results have identified p11 as a key molecule in a specific cell type that regulates stress-induced depression, which provides a framework for the development of new strategies to treat stress-associated mental illnesses.

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer.

PubMed

Mason, Jacqueline M; Wei, Xin; Fletcher, Graham C; Kiarash, Reza; Brokx, Richard; Hodgson, Richard; Beletskaya, Irina; Bray, Mark R; Mak, Tak W

2017-03-21

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 K i = 0.09 ± 0.02 nM; cellular Mps1 EC 50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors.

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer

PubMed Central

Mason, Jacqueline M.; Wei, Xin; Fletcher, Graham C.; Kiarash, Reza; Brokx, Richard; Hodgson, Richard; Beletskaya, Irina; Bray, Mark R.; Mak, Tak W.

2017-01-01

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors. PMID:28270606

Cyclophilin B facilitates the replication of Orf virus.

PubMed

Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

2017-06-15

Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID 50 ) assay and qRT-PCR detection. In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

Cellular transport of l-arginine determines renal medullary blood flow in control rats, but not in diabetic rats despite enhanced cellular uptake capacity.

PubMed

Persson, Patrik; Fasching, Angelica; Teerlink, Tom; Hansell, Peter; Palm, Fredrik

2017-02-01

Diabetes mellitus is associated with decreased nitric oxide bioavailability thereby affecting renal blood flow regulation. Previous reports have demonstrated that cellular uptake of l-arginine is rate limiting for nitric oxide production and that plasma l-arginine concentration is decreased in diabetes. We therefore investigated whether regional renal blood flow regulation is affected by cellular l-arginine uptake in streptozotocin-induced diabetic rats. Rats were anesthetized with thiobutabarbital, and the left kidney was exposed. Total, cortical, and medullary renal blood flow was investigated before and after renal artery infusion of increasing doses of either l-homoarginine to inhibit cellular uptake of l-arginine or N ω -nitro- l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase. l-Homoarginine infusion did not affect total or cortical blood flow in any of the groups, but caused a dose-dependent reduction in medullary blood flow. l-NAME decreased total, cortical and medullary blood flow in both groups. However, the reductions in medullary blood flow in response to both l-homoarginine and l-NAME were more pronounced in the control groups compared with the diabetic groups. Isolated cortical tubular cells displayed similar l-arginine uptake capacity whereas medullary tubular cells isolated from diabetic rats had increased l-arginine uptake capacity. Diabetics had reduced l-arginine concentrations in plasma and medullary tissue but increased l-arginine concentration in cortical tissue. In conclusion, the reduced l-arginine availability in plasma and medullary tissue in diabetes results in reduced nitric oxide-mediated regulation of renal medullary hemodynamics. Cortical blood flow regulation displays less dependency on extracellular l-arginine and the upregulated cortical tissue l-arginine may protect cortical hemodynamics in diabetes. Copyright © 2017 the American Physiological Society.

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes.

PubMed

Alam, Shafiul; Abdullah, Chowdhury S; Aishwarya, Richa; Orr, A Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B; Bhuiyan, Md Shenuarin

2017-08-31

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. © 2017 The Author(s).

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

PubMed Central

Alam, Shafiul; Abdullah, Chowdhury S.; Aishwarya, Richa; Orr, A. Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B.

2017-01-01

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. PMID:28667101

Cell migration, intercalation and growth regulate mammalian cochlear extension.

PubMed

Driver, Elizabeth Carroll; Northrop, Amy; Kelley, Matthew W

2017-10-15

Developmental remodeling of the sensory epithelium of the cochlea is required for the formation of an elongated, tonotopically organized auditory organ, but the cellular processes that mediate these events are largely unknown. We used both morphological assessments of cellular rearrangements and time-lapse imaging to visualize cochlear remodeling in mouse. Analysis of cell redistribution showed that the cochlea extends through a combination of radial intercalation and cell growth. Live imaging demonstrated that concomitant cellular intercalation results in a brief period of epithelial convergence, although subsequent changes in cell size lead to medial-lateral spreading. Supporting cells, which retain contact with the basement membrane, exhibit biased protrusive activity and directed movement along the axis of extension. By contrast, hair cells lose contact with the basement membrane, but contribute to continued outgrowth through increased cell size. Regulation of cellular protrusions, movement and intercalation within the cochlea all require myosin II. These results establish, for the first time, many of the cellular processes that drive the distribution of sensory cells along the tonotopic axis of the cochlea. © 2017. Published by The Company of Biologists Ltd.

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

PubMed Central

Rhein, Cosima; Tripal, Philipp; Seebahn, Angela; Konrad, Alice; Kramer, Marcel; Nagel, Christine; Kemper, Jonas; Bode, Jens; Mühle, Christiane; Gulbins, Erich; Reichel, Martin; Becker, Cord-Michael; Kornhuber, Johannes

2012-01-01

Background Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. Methodology/Principal Findings We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. Conclusions/Significance These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity. PMID:22558155

Cellular Bases of Light-regulated Gravity Responses

NASA Technical Reports Server (NTRS)

Roux, Stanley J.

2003-01-01

This report summarizes the most significant research accomplished in our NAG2-1347 project on the cellular bases of light-regulated gravity responses, It elaborates mainly on our discovery of the role of calcium currents in gravity-directed polar development in single germinating spore cells of the fern Ceratopteris, our development of RNA silencing as a viable method of suppressing the expression of specific genes in Ceratopteris, and on the structure, expression and distribution of members of the annexin family in flowering plants, especially Arabidopsis.

A New Paradigm for Ovarian Sex Cord-Stromal Tumor Development

DTIC Science & Technology

2017-05-01

Transforming growth factor beta (TGFB) family members regulate multiple cellular functions and key reproductive processes in a contextually dependent manner...Appendices……………………………………………………………11 4 1. Introduction Transforming growth factor beta (TGFβ) family members regulate a myriad of cellular functions and... transformation 3. Accomplishments  What were the major goals of the project? The major goal during this reporting period is to identify the oncogenic

A uniquely adaptable pore is consistent with NALCN being an ion sensor

PubMed Central

Senatore, Adriano; Spafford, J. David

2013-01-01

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN’s most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd3+-sensitive, NMDG+-impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (ILP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Nax or Cav1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels. PMID:23442378

A uniquely adaptable pore is consistent with NALCN being an ion sensor.

PubMed

Senatore, Adriano; Spafford, J David

2013-01-01

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN's most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd ( 3+) -sensitive, NMDG (+) -impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (ILP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Nax or Cav 1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels.

Regulation of raft-dependent endocytosis

PubMed Central

Lajoie, P; Nabi, IR

2007-01-01

Abstract Raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrin-independent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveo-lae, smooth plasmalemmal vesicles that form a subdomain of cholesterol and sphingolipid-rich lipid rafts and that are enriched for caveolin-1. While sharing common mechanisms, like cholesterol sensitivity, raft endocytic routes show differential regulation by various cellular components including caveolin-1, dynamin-2 and regulators of the actin cytoskeleton. Dynamin-dependent raft pathways, mediated by caveolae and morphologically equivalent non-caveolin vesicular intermediates, are referred to as caveolae/raft-dependent endocytosis. In contrast, dynamin-independent raft pathways are mediated by non-caveolar intermediates. Raft-dependent endocytosis is regulated by tyrosine kinase inhibitors and, through the regulation of the internalization of various ligands, receptors and effectors, is also a determinant of cellular signaling. In this review, we characterize and discuss the regulation of raft-dependent endocytic pathways and the role of key regulators such as caveolin-1. PMID:17760830

Wrecked regulation of intrinsically disordered proteins in diseases: pathogenicity of deregulated regulators

PubMed Central

Uversky, Vladimir N.

2014-01-01

Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs) are typically related to regulation, signaling, and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases. PMID:25988147

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

PubMed

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-02-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells

PubMed Central

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-01-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer. PMID:25692008

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

PubMed

Nahta, Rita; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Andrade-Vieira, Rafaela; Bay, Sarah N; Brown, Dustin G; Calaf, Gloria M; Castellino, Robert C; Cohen-Solal, Karine A; Colacci, Annamaria; Cruickshanks, Nichola; Dent, Paul; Di Fiore, Riccardo; Forte, Stefano; Goldberg, Gary S; Hamid, Roslida A; Krishnan, Harini; Laird, Dale W; Lasfar, Ahmed; Marignani, Paola A; Memeo, Lorenzo; Mondello, Chiara; Naus, Christian C; Ponce-Cusi, Richard; Raju, Jayadev; Roy, Debasish; Roy, Rabindra; Ryan, Elizabeth P; Salem, Hosni K; Scovassi, A Ivana; Singh, Neetu; Vaccari, Monica; Vento, Renza; Vondráček, Jan; Wade, Mark; Woodrick, Jordan; Bisson, William H

2015-06-01

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Interplay of autophagy, receptor tyrosine kinase signalling and endocytic trafficking

PubMed Central

Fraser, Jane; Cabodevilla, Ainara G.; Simpson, Joanne; Gammoh, Noor

2017-01-01

Vesicular trafficking events play key roles in the compartmentalization and proper sorting of cellular components. These events have crucial roles in sensing external signals, regulating protein activities and stimulating cell growth or death decisions. Although mutations in vesicle trafficking players are not direct drivers of cellular transformation, their activities are important in facilitating oncogenic pathways. One such pathway is the sensing of external stimuli and signalling through receptor tyrosine kinases (RTKs). The regulation of RTK activity by the endocytic pathway has been extensively studied. Compelling recent studies have begun to highlight the association between autophagy and RTK signalling. The influence of this interplay on cellular status and its relevance in disease settings will be discussed here. PMID:29233871

Inherent X-Linked Genetic Variability and Cellular Mosaicism Unique to Females Contribute to Sex-Related Differences in the Innate Immune Response.

PubMed

Spolarics, Zoltan; Peña, Geber; Qin, Yong; Donnelly, Robert J; Livingston, David H

2017-01-01

Females have a longer lifespan and better general health than males. Considerable number of studies also demonstrated that, after trauma and sepsis, females present better outcomes as compared to males indicating sex-related differences in the innate immune response. The current notion is that differences in the immuno-modulatory effects of sex hormones are the underlying causative mechanism. However, the field remains controversial and the exclusive role of sex hormones has been challenged. Here, we propose that polymorphic X-linked immune competent genes, which are abundant in the population are important players in sex-based immuno-modulation and play a key role in causing sex-related outcome differences following trauma or sepsis. We describe the differences in X chromosome (ChrX) regulation between males and females and its consequences in the context of common X-linked polymorphisms at the individual as well as population level. We also discuss the potential pathophysiological and immune-modulatory aspects of ChrX cellular mosaicism, which is unique to females and how this may contribute to sex-biased immune-modulation. The potential confounding effects of ChrX skewing of cell progenitors at the bone marrow is also presented together with aspects of acute trauma-induced de novo ChrX skewing at the periphery. In support of the hypothesis, novel observations indicating ChrX skewing in a female trauma cohort as well as case studies depicting the temporal relationship between trauma-induced cellular skewing and the clinical course are also described. Finally, we list and discuss a selected set of polymorphic X-linked genes, which are frequent in the population and have key regulatory or metabolic functions in the innate immune response and, therefore, are primary candidates for mediating sex-biased immune responses. We conclude that sex-related differences in a variety of disease processes including the innate inflammatory response to injury and infection may be related to the abundance of X-linked polymorphic immune-competent genes, differences in ChrX regulation, and inheritance patterns between the sexes and the presence of X-linked cellular mosaicism, which is unique to females.

The Emerging Role of Skeletal Muscle Metabolism as a Biological Target and Cellular Regulator of Cancer-Induced Muscle Wasting

PubMed Central

Carson, James A.; Hardee, Justin P.; VanderVeen, Brandon N.

2015-01-01

While skeletal muscle mass is an established primary outcome related to understanding cancer cachexia mechanisms, considerable gaps exist in our understanding of muscle biochemical and functional properties that have recognized roles in systemic health. Skeletal muscle quality is a classification beyond mass, and is aligned with muscle’s metabolic capacity and substrate utilization flexibility. This supplies an additional role for the mitochondria in cancer-induced muscle wasting. While the historical assessment of mitochondria content and function during cancer-induced muscle loss was closely aligned with energy flux and wasting susceptibility, this understanding has expanded to link mitochondria dysfunction to cellular processes regulating myofiber wasting. The primary objective of this article is to highlight muscle mitochondria and oxidative metabolism as a biological target of cancer cachexia and also as a cellular regulator of cancer-induced muscle wasting. Initially, we examine the role of muscle metabolic phenotype and mitochondria content in cancer-induced wasting susceptibility. We then assess the evidence for cancer-induced regulation of skeletal muscle mitochondrial biogenesis, dynamics, mitophagy, and oxidative stress. In addition, we discuss environments associated with cancer cachexia that can impact the regulation of skeletal muscle oxidative metabolism. The article also examines the role of cytokine-mediated regulation of mitochondria function regulation, followed by the potential role of cancer-induced hypogonadism. Lastly, a role for decreased muscle use in cancer-induced mitochondrial dysfunction is reviewed. PMID:26593326

Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

PubMed

Jain, Shruti; Bhattacharyya, Kausik; Bakshi, Rachit; Narang, Ankita; Brahmachari, Vani

2017-04-01

The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Cellular and laminar expression of Dab-1 during the postnatal critical period in cat visual cortex and the effects of dark rearing.

PubMed

Kiser, Paul J; Liu, Zijing; Wilt, Steven D; Mower, George D

2011-04-06

This study describes postnatal critical period changes in cellular and laminar expression of Dab-1, a gene shown to play a role in controlling neuronal positioning during embryonic brain development, in cat visual cortex and the effects of dark rearing (DR). At 1week, there is dense cellular staining which is uniform across cortical layers and very light neuropil staining. At the peak of the critical period (5weeks), dense cell staining is largely restricted to large pyramidal cells of deep layer III and layer V, there is faint cell body staining throughout all cortical layers, neuropil staining is markedly increased and uniform in layers III to VI. This dramatic change in laminar and cellular labeling is independent of visual input, since immunostaining is similar in 5-week DR cats. By 10weeks, the mature laminar and cellular staining pattern is established and the major subsequent change is a further reduction in the density of cellular staining in all cortical layers. Neuropil staining is pronounced and uniform across cortical layers. These developmental changes are altered by DR. Quantification by cell counts indicated that age and DR interact such that differences in cellular expression are opposite in direction between 5- and 20-week-old cats. This bidirectional regulation of cellular expression is the same in all cortical laminae. The bidirectional regulation of cellular expression matches the effects of age and DR on physiological plasticity during the critical period as assessed by ocular dominance shifts in response to monocular deprivation. Copyright © 2011 Elsevier B.V. All rights reserved.

Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells.

PubMed

Foresti, Roberta; Bains, Sandip K; Pitchumony, Tamil Selvi; de Castro Brás, Lisandra E; Drago, Filippo; Dubois-Randé, Jean-Luc; Bucolo, Claudio; Motterlini, Roberto

2013-10-01

The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5-20 μM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hydrogen peroxide production is affected by oxygen levels in mammalian cell culture.

PubMed

Maddalena, Lucas A; Selim, Shehab M; Fonseca, Joao; Messner, Holt; McGowan, Shannon; Stuart, Jeffrey A

2017-11-04

Although oxygen levels in the extracellular space of most mammalian tissues are just a few percent, under standard cell culture conditions they are not regulated and are often substantially higher. Some cellular sources of reactive oxygen species, like NADPH oxidase 4, are sensitive to oxygen levels in the range between 'normal' physiological (typically 1-5%) and standard cell culture (up to 18%). Hydrogen peroxide in particular participates in signal transduction pathways via protein redox modifications, so the potential increase in its production under standard cell culture conditions is important to understand. We measured the rates of cellular hydrogen peroxide production in some common cell lines, including C2C12, PC-3, HeLa, SH-SY5Y, MCF-7, and mouse embryonic fibroblasts (MEFs) maintained at 18% or 5% oxygen. In all instances the rate of hydrogen peroxide production by these cells was significantly greater at 18% oxygen than at 5%. The increase in hydrogen peroxide production at higher oxygen levels was either abolished or substantially reduced by treatment with GKT 137831, a selective inhibitor of NADPH oxidase subunits 1 and 4. These data indicate that oxygen levels experienced by cells in culture influence hydrogen peroxide production via NADPH oxidase 1/4, highlighting the importance of regulating oxygen levels in culture near physiological values. However, we measured pericellular oxygen levels adjacent to cell monolayers under a variety of conditions and with different cell lines and found that, particularly when growing at 5% incubator oxygen levels, pericellular oxygen was often lower and variable. Together, these observations indicate the importance, and difficulty, of regulating oxygen levels experienced by cells in culture. Copyright © 2017 Elsevier Inc. All rights reserved.

p70 ribosomal S6 kinase regulates subpleural fibrosis following transforming growth factor-α expression in the lung

PubMed Central

Madala, Satish K.; Thomas, George; Edukulla, Ramakrishna; Davidson, Cynthia; Schmidt, Stephanie; Schehr, Angelica

2015-01-01

The p70 ribosomal S6 kinase (S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with fibrogenesis. Recent studies demonstrate that aberrant mTORC1-S6K signaling contributes to various pathological conditions, but a direct role in pulmonary fibroproliferation has not been established. Increased phosphorylation of the S6K pathway is detected immediately following transforming growth factor-α (TGF-α) expression in a transgenic model of progressive lung fibrosis. To test the hypothesis that the S6K directly regulates pulmonary fibroproliferative disease we determined the cellular sites of S6K phosphorylation during the induction of fibrosis in the TGF-α model and tested the efficacy of specific pharmacological inhibition of the S6K pathway to prevent and reverse fibrotic disease. Following TGF-α expression increased phosphorylation of the S6K was detected in the airway and alveolar epithelium and the mesenchyme of advanced subpleural fibrotic regions. Specific inhibition of the S6K with the small molecule inhibitor LY-2584702 decreased TGF-α and platelet-derived growth factor-β-induced proliferation of lung fibroblasts in vitro. Administration of S6K inhibitors to TGF-α mice prevented the development of extensive subpleural fibrosis and alterations in lung mechanics, and attenuated the increase in total lung hydroxyproline. S6K inhibition after fibrosis was established attenuated the progression of subpleural fibrosis. Together these studies demonstrate targeting the S6K pathway selectively modifies the progression of pulmonary fibrosis in the subpleural compartment of the lung. PMID:26566903

Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins*

PubMed Central

Medina, Frank; Carter, Angela M.; Dada, Olugbenga; Gutowski, Stephen; Hadas, Jana; Chen, Zhe; Sternweis, Paul C.

2013-01-01

The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs. PMID:23493395

Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.

PubMed

Goris, Marianne; Magin, Robert S; Foyn, Håvard; Myklebust, Line M; Varland, Sylvia; Ree, Rasmus; Drazic, Adrian; Bhambra, Parminder; Støve, Svein I; Baumann, Markus; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

2018-04-24

N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties. Copyright © 2018 the Author(s). Published by PNAS.

Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents.

PubMed

Kita, Kayoko; Suzuki, Toshihide; Ochi, Takafumi

2007-05-01

In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments.

Calmodulin-like protein 3 is an estrogen receptor alpha coregulator for gene expression and drug response in a SNP, estrogen, and SERM-dependent fashion.

PubMed

Qin, Sisi; Ingle, James N; Liu, Mohan; Yu, Jia; Wickerham, D Lawrence; Kubo, Michiaki; Weinshilboum, Richard M; Wang, Liewei

2017-08-18

We previously performed a case-control genome-wide association study in women treated with selective estrogen receptor modulators (SERMs) for breast cancer prevention and identified single nucleotide polymorphisms (SNPs) in ZNF423 as potential biomarkers for response to SERM therapy. The ZNF423rs9940645 SNP, which is approximately 200 bp away from the estrogen response elements, resulted in the SNP, estrogen, and SERM-dependent regulation of ZNF423 expression and, "downstream", that of BRCA1. Electrophoretic mobility shift assay-mass spectrometry was performed to identify proteins binding to the ZNF423 SNP and coordinating with estrogen receptor alpha (ERα). Clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing was applied to generate ZR75-1 breast cancer cells with different ZNF423 SNP genotypes. Both cultured cells and mouse xenograft models with different ZNF423 SNP genotypes were used to study the cellular responses to SERMs and poly(ADP-ribose) polymerase (PARP) inhibitors. We identified calmodulin-like protein 3 (CALML3) as a key sensor of this SNP and a coregulator of ERα, which contributes to differential gene transcription regulation in an estrogen and SERM-dependent fashion. Furthermore, using CRISPR/Cas9-engineered ZR75-1 breast cancer cells with different ZNF423 SNP genotypes, striking differences in cellular responses to SERMs and PARP inhibitors, alone or in combination, were observed not only in cells but also in a mouse xenograft model. Our results have demonstrated the mechanism by which the ZNF423 rs9940645 SNP might regulate gene expression and drug response as well as its potential role in achieving more highly individualized breast cancer therapy.

Regulation of the ErbB network by the MIG6 feedback loop in physiology, tumor suppression and responses to oncogene-targeted therapeutics.

PubMed

Anastasi, Sergio; Lamberti, Dante; Alemà, Stefano; Segatto, Oreste

2016-02-01

The ErbB signaling network instructs the execution of key cellular programs, such as cell survival, proliferation and motility, through the generation of robust signals of defined strength and duration. In contrast, unabated ErbB signaling disrupts tissue homeostasis and leads to cell transformation. Cells oppose the threat inherent in excessive ErbB activity through several mechanisms of negative feedback regulation. Inducible feedback inhibitors (IFIs) are expressed in the context of transcriptional responses triggered by ErbB signaling, thus being uniquely suited to regulate ErbB activity during the execution of complex cellular programs. This review focuses on MIG6, an IFI that restrains ErbB signaling by mediating ErbB kinase suppression and receptor down-regulation. We will review key issues in MIG6 function, regulation and tumor suppressor activity. Subsequently, the role for MIG6 loss in the pathogenesis of tumors driven by ErbB oncogenes as well as in the generation of cellular addiction to ErbB signaling will be discussed. We will conclude by analyzing feedback inhibition by MIG6 in the context of therapies directed against ErbB and non-ErbB oncogenes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fabrication and characterisation of a fully auxetic 3D lattice structure via selective electron beam melting

NASA Astrophysics Data System (ADS)

Warmuth, Franziska; Osmanlic, Fuad; Adler, Lucas; Lodes, Matthias A.; Körner, Carolin

2017-02-01

A three-dimensional fully auxetic cellular structure with negative Poisson’s ratio is presented. Samples are fabricated from Ti6Al4V powder via selective electron beam melting. The influence of the strut thickness and the amplitude of the strut on the mechanical properties and the deformation behaviour of cellular structures is studied.

Amadori-glycated phosphatidylethanolamine enhances the physical stability and selective targeting ability of liposomes

PubMed Central

Miyazawa, Taiki; Kamiyoshihara, Reina; Shimizu, Naoki; Harigae, Takahiro; Otoki, Yurika; Ito, Junya; Kato, Shunji; Miyazawa, Teruo

2018-01-01

Liposomes consisting of 100% phosphatidylcholine exhibit poor membrane fusion, cellular uptake and selective targeting capacities. To overcome these limitations, we used Amadori-glycated phosphatidylethanolamine, which is universally present in animals and commonly consumed in foods. We found that liposomes containing Amadori-glycated phosphatidylethanolamine exhibited significantly reduced negative membrane potential and demonstrated high cellular uptake. PMID:29515844

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

PubMed

Herlo, Rasmus; Lund, Viktor K; Lycas, Matthew D; Jansen, Anna M; Khelashvili, George; Andersen, Rita C; Bhatia, Vikram; Pedersen, Thomas S; Albornoz, Pedro B C; Johner, Niklaus; Ammendrup-Johnsen, Ina; Christensen, Nikolaj R; Erlendsson, Simon; Stoklund, Mikkel; Larsen, Jannik B; Weinstein, Harel; Kjærulff, Ole; Stamou, Dimitrios; Gether, Ulrik; Madsen, Kenneth L

2018-05-15

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Determinants of orofacial clefting I: Effects of 5-Aza-2'-deoxycytidine on cellular processes and gene expression during development of the first branchial arch.

PubMed

Mukhopadhyay, Partha; Seelan, Ratnam S; Rezzoug, Francine; Warner, Dennis R; Smolenkova, Irina A; Brock, Guy; Pisano, M Michele; Greene, Robert M

2017-01-01

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect. Copyright © 2016 Elsevier Inc. All rights reserved.

Shifts in growth strategies reflect tradeoffs in cellular economics

PubMed Central

Molenaar, Douwe; van Berlo, Rogier; de Ridder, Dick; Teusink, Bas

2009-01-01

The growth rate-dependent regulation of cell size, ribosomal content, and metabolic efficiency follows a common pattern in unicellular organisms: with increasing growth rates, cell size and ribosomal content increase and a shift to energetically inefficient metabolism takes place. The latter two phenomena are also observed in fast growing tumour cells and cell lines. These patterns suggest a fundamental principle of design. In biology such designs can often be understood as the result of the optimization of fitness. Here we show that in basic models of self-replicating systems these patterns are the consequence of maximizing the growth rate. Whereas most models of cellular growth consider a part of physiology, for instance only metabolism, the approach presented here integrates several subsystems to a complete self-replicating system. Such models can yield fundamentally different optimal strategies. In particular, it is shown how the shift in metabolic efficiency originates from a tradeoff between investments in enzyme synthesis and metabolic yields for alternative catabolic pathways. The models elucidate how the optimization of growth by natural selection shapes growth strategies. PMID:19888218

Control of neuronal excitability by Group I metabotropic glutamate receptors.

PubMed

Correa, Ana Maria Bernal; Guimarães, Jennifer Diniz Soares; Dos Santos E Alhadas, Everton; Kushmerick, Christopher

2017-10-01

Metabotropic glutamate (mGlu) receptors couple through G proteins to regulate a large number of cell functions. Eight mGlu receptor isoforms have been cloned and classified into three Groups based on sequence, signal transduction mechanisms and pharmacology. This review will focus on Group I mGlu receptors, comprising the isoforms mGlu 1 and mGlu 5 . Activation of these receptors initiates both G protein-dependent and -independent signal transduction pathways. The G-protein-dependent pathway involves mainly Gα q , which can activate PLCβ, leading initially to the formation of IP 3 and diacylglycerol. IP 3 can release Ca 2+ from cellular stores resulting in activation of Ca 2+ -dependent ion channels. Intracellular Ca 2+ , together with diacylglycerol, activates PKC, which has many protein targets, including ion channels. Thus, activation of the G-protein-dependent pathway affects cellular excitability though several different effectors. In parallel, G protein-independent pathways lead to activation of non-selective cationic currents and metabotropic synaptic currents and potentials. Here, we provide a survey of the membrane transport proteins responsible for these electrical effects of Group I metabotropic glutamate receptors.

Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

PubMed

Lamarche, M J; Borawski, J; Bose, A; Capacci-Daniel, C; Colvin, R; Dennehy, M; Ding, J; Dobler, M; Drumm, J; Gaither, L A; Gao, J; Jiang, X; Lin, K; McKeever, U; Puyang, X; Raman, P; Thohan, S; Tommasi, R; Wagner, K; Xiong, X; Zabawa, T; Zhu, S; Wiedmann, B

2012-10-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

Anti-Hepatitis C Virus Activity and Toxicity of Type III Phosphatidylinositol-4-Kinase Beta Inhibitors

PubMed Central

LaMarche, M. J.; Borawski, J.; Bose, A.; Capacci-Daniel, C.; Colvin, R.; Dennehy, M.; Ding, J.; Dobler, M.; Drumm, J.; Gaither, L. A.; Gao, J.; Jiang, X.; Lin, K.; McKeever, U.; Puyang, X.; Raman, P.; Thohan, S.; Tommasi, R.; Wagner, K.; Xiong, X.; Zabawa, T.; Zhu, S.

2012-01-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C. PMID:22825118

Generation of a Tet-On Expression System to Study Transactivation Ability of Tax-2.

PubMed

Bignami, Fabio; Sozzi, Riccardo Alessio; Pilotti, Elisabetta

2017-01-01

HTLV Tax proteins (Tax-1 and Tax-2) are known to be able to transactivate several host cellular genes involved in complex molecular pathways. Here, we describe a stable and regulated high-level expression model based on Tet-On system, to study the capacity of Tax-2 to transactivate host genes. In particular, the Jurkat Tet-On cell line suitable for evaluating the ability of Tax-2 to stimulate transactivation of a specific host gene, CCL3L1 (C-C motif chemokine ligand 3 like 1 gene), was selected. Then, a plasmid expressing tax-2 gene under control of a tetracycline-response element was constructed. To avoid the production of a fusion protein between the report gene and the inserted gene, a bidirectional plasmid was designed. Maximum expression and fast response time were achieved by using nucleofection technology as transfection method. After developing an optimized protocol for efficiently transferring tax-2 gene in Jurkat Tet-On cellular model and exposing transfected cells to Dox (doxycycline, a tetracycline derivate), a kinetics of tax-2 expression through TaqMan Real-time PCR assay was determined.

The ML1Nx2 Phosphatidylinositol 3,5-Bisphosphate Probe Shows Poor Selectivity in Cells.

PubMed

Hammond, Gerald R V; Takasuga, Shunsuke; Sasaki, Takehiko; Balla, Tamas

2015-01-01

Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action. In this study, we have evaluated a recently reported PtdIns(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. We report that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PtdIns(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid's synthesis. We therefore conclude that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PtdIns(3,5)P2.

Molecular imaging of labile iron(II) pools in living cells with a turn-on fluorescent probe.

PubMed

Au-Yeung, Ho Yu; Chan, Jefferson; Chantarojsiri, Teera; Chang, Christopher J

2013-10-09

Iron is an essential metal for living organisms, but misregulation of its homeostasis at the cellular level can trigger detrimental oxidative and/or nitrosative stress and damage events. Motivated to help study the physiological and pathological consequences of biological iron regulation, we now report a reaction-based strategy for monitoring labile Fe(2+) pools in aqueous solution and living cells. Iron Probe 1 (IP1) exploits a bioinspired, iron-mediated oxidative C-O bond cleavage reaction to achieve a selective turn-on response to Fe(2+) over a range of cellular metal ions in their bioavailable forms. We show that this first-generation chemical tool for fluorescence Fe(2+) detection can visualize changes in exchangeable iron stores in living cells upon iron supplementation or depletion, including labile iron pools at endogenous, basal levels. Moreover, IP1 can be used to identify reversible expansion of labile iron pools by stimulation with vitamin C or the iron regulatory hormone hepcidin, providing a starting point for further investigations of iron signaling and stress events in living systems as well as future probe development.

Bisubstrate inhibitors of protein kinases: from principle to practical applications.

PubMed

Lavogina, Darja; Enkvist, Erki; Uri, Asko

2010-01-01

Bisubstrate inhibitors consist of two conjugated fragments, each targeted to a different binding site of a bisubstrate enzyme. The design of bisubstrate inhibitors presupposes the formation of the ternary complex in the course of the catalyzed reaction. The principle advantage of bisubstrate inhibitors is their ability to generate more interactions with the target enzyme that could result in improved affinity and selectivity of the conjugates, when compared with single-site inhibitors. Among phosphotransferases, the approach was first successfully used for adenylate kinase in 1973. Since then, several types of bisubstrate inhibitors have been developed for protein kinases, including conjugates of peptides with nucleotides, adenosine derivatives and potent ATP-competitive inhibitors. Earlier bisubstrate inhibitors had pharmacokinetic qualities that were unsuitable for cellular experiments and hence were mostly used for in vitro studies. The recently constructed conjugates of adenosine derivatives and D-arginine-rich peptides (ARCs) possess high kinase affinity, high biological and chemical stability and good cell plasma membrane penetrative properties that enable their application in the regulation of cellular protein phosphorylation balances in cell and tissue experiments.

Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

PubMed

Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

2013-12-01

Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

Modeling the cost and benefit of proteome regulation in a growing bacterial cell

NASA Astrophysics Data System (ADS)

Sharma, Pooja; Pratim Pandey, Parth; Jain, Sanjay

2018-07-01

Escherichia coli cells differentially regulate the production of metabolic and ribosomal proteins in order to stay close to an optimal growth rate in different environments, and exhibit the bacterial growth laws as a consequence. We present a simple mathematical model of a growing-dividing cell in which an internal dynamical mechanism regulates the allocation of proteomic resources between different protein sectors. The model allows an endogenous determination of the growth rate of the cell as a function of cellular and environmental parameters, and reproduces the bacterial growth laws. We use the model and its variants to study the balance between the cost and benefit of regulation. A cost is incurred because cellular resources are diverted to produce the regulatory apparatus. We show that there is a window of environments or a ‘niche’ in which the unregulated cell has a higher fitness than the regulated cell. Outside this niche there is a large space of constant and time varying environments in which regulation is an advantage. A knowledge of the ‘niche boundaries’ allows one to gain an intuitive understanding of the class of environments in which regulation is an advantage for the organism and which would therefore favour the evolution of regulation. The model allows us to determine the ‘niche boundaries’ as a function of cellular parameters such as the size of the burden of the regulatory apparatus. This class of models may be useful in elucidating various tradeoffs in cells and in making in-silico predictions relevant for synthetic biology.