Chemical Modulation of Endocytic Sorting Augments Adeno-Associated Viral Transduction [Cell Biology]

November 2nd, 2015 by Berry, G. E., Asokan, A.

Intracellular trafficking of viruses can be influenced by a variety of inter-connected cellular sorting and degradation pathways involving endo-lysosomal vesicles, the ubiquitin-proteasome system, autophagy-based or ER-associated machinery. In case of recombinant adeno-associated viruses (AAV), proteasome inhibitors are known to prevent degradation of ubiquitinated AAV capsids, thereby leading to increased nuclear accumulation and transduction. However, the impact of other cellular degradation pathways on AAV trafficking is not well-understood. In the current report, we screened a panel of small molecules focused on modulating different cellular degradation pathways and identified Eeyarestatin I (EerI) as a novel reagent that enhances AAV transduction. EerI improved AAV transduction by an order of magnitude regardless of vector dose, genome architecture, cell type, or serotype. This effect was preceded by sequestration of AAV within enlarged vesicles that were dispersed throughout the cytoplasm. Specifically, EerI treatment redirected AAV particles towards large vesicles positive for late endosomal (Rab7) and lysosomal (LAMP1) markers. Notably, MG132 and EerI (proteasomal and endoplasmic reticulum-associated degradation (ERAD) inhibitors, respectively) appear to enhance AAV transduction by increasing the intracellular accumulation of viral particles in a mutually exclusive fashion. Taken together, our results expand on potential strategies to redirect recombinant AAV vectors towards more productive trafficking pathways by deregulating cellular degradation mechanisms.

Structural basis of Ribosomal S6 Kinase 1 (RSK1) inhibition by S100B Protein: modulation of the Extracellular Signal-regulated Kinase (ERK) signaling cascade in a calcium-dependent way [Protein Structure and Folding]

November 2nd, 2015 by

Mitogen-activated protein kinases (MAPK) promote MAPK activated protein kinase (MAPKAPK) activation. In the MAPK pathway responsible to cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by calcium-binding S100 proteins in malignant melanomas. Here we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The calcium-dependent binding of S100B to the calcium/calmodulin dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent to the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca2+ binding proteins could influence the activity of CaMK domain containing protein kinases. Our crystallographic, small angle X-ray scattering (SAXS) and NMR analysis revealed that S100B forms a ″fuzzy″ complex with RSK1 peptide ligands. Based on fast-kinetics experiments we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.
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Integrin {alpha}V{beta}5-mediated removal of apoptotic cell debris by the eye lens and its inhibition by UV-light exposure [Cell Biology]

November 2nd, 2015 by Chauss, D., Brennan, L. A., Bakina, O., Kantorow, M.

Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important towards establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV-light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV-light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high-throughput RNA sequencing and bioinformatics analysis we establish that lens epithelial cells express members of the integrin αVβ5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVβ5 and with RAB7 and RILP that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVβ5 receptor is required for lens epithelial cell phagocytosis and that UV-light treatment of lens epithelial cells results in damage to the αVβ5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVβ5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.

Regulation of Focal Adhesion Dynamics and Cell Motility by EB2 and Hax1 Complex [Signal Transduction]

November 2nd, 2015 by Liu, H., Yue, J., Huang, H., Gou, X., Chen, S.-Y., Zhao, Y., Wu, X.

Cell migration is a fundamental cellular process, requiring integrated activities of cytoskeleton, membrane, and cell/ECM adhesions. Many cytoskeletal activities rely on microtubule filaments. It has been speculated that microtubules can serve as tracks to deliver proteins essential for focal adhesion turnover. Three microtubule end-binding proteins (EB1, EB2 and EB3) in mammalian cells can track the plus ends of growing microtubules. EB1 and EB3 together can regulate microtubule dynamics by promoting microtubule growth and suppressing catastrophe; while in contrast, EB2 does not play a direct role in microtubule dynamic instability, and little is known about EB2 cellular function. By quantitative proteomics, we identified mammalian HAX1 (HCLS1 associated protein X-1), which associates with EB2 specifically. Knockdown of HAX1 and EB2 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration in vitro and in vivo. Our results further demonstrate that cell motility and focal adhesion turnover requires interaction between Hax1 and EB2. Together, our findings provide new insights for this critical cellular process, suggesting that EB2 association with Hax1 plays a significant role in focal adhesion turnover and epidermal migration.

Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B) [Enzymology]

November 1st, 2015 by Haigney, A., Ricketts, M. D., Marmorstein, R.

The HAT-B enzyme complex is responsible for acetylating newly synthesized H4 on lysines K5 and K12. HAT-B is a multi subunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2 and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and independent chromatin assembly. In order to elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2 and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions are not sufficient for this enhanced activity. Based on this data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

Inhibition of mitochondrial complex II by the anti-cancer agent lonidamine [Lipids]

October 31st, 2015 by

The anti-tumor agent Lonidamine (LND, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that significant amount of succinate is accumulated in LND treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase (SQR) activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species (ROS) through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (HYaluronan Binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts. [Metabolism]

October 30th, 2015 by

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We have recently reported that HYBID (HYaluronan Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, bFGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression, but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower-molecular-weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, bFGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high-molecular-weight HA by HAS up-regulation and HYBID down-regulation, and suggest that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.
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Telomerase deficiency causes alveolar stem cell senescence-associated low-grade inflammation in lungs [Molecular Bases of Disease]

October 30th, 2015 by

Mutations of human telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) are associated with a subset of lung aging diseases, but the mechanisms by which TERC and TERT participate in lung diseases remain unclear. In this report, we show that knockout (KO) of the mouse gene TERC or TERT causes pulmonary alveolar stem cell replicative senescence, epithelial impairment, formation of alveolar sacs, and characteristic inflammatory phenotype. Deficiency in TERC or TERT causes a remarkable elevation in various proinflammatory cytokines, including IL-1, IL-6, CXCL15 (human IL-8 homolog), IL-10, TNF-α and monocyte chemotactic protein 1 (chemokine ligand 2, CCL2), decrease in TGF-β1 and TGFβRI receptor in the lungs, and spillover of IL-6 and CXCL15 into the bronchoalveolar lavage fluids. In addition to increased gene expressions of α-smooth muscle actin (α-SMA) and collagen 1α1 (Col1α1) suggesting myofibroblast differentiation, TERC deficiency also leads to marked cellular infiltrations of a mononuclear cell population positive for the leukocyte common antigen CD45 (LCA), low-affinity Fc receptor CD16/CD32, and pattern recognition receptor CD11b in the lungs. Our data demonstrate for the first time that telomerase deficiency triggers alveolar stem cell replicative senescence-associated low-grade inflammation, thereby driving pulmonary premature aging, alveolar sac formation and fibrotic lesion.

{alpha}-Defensins induce a post-translational modification of LDL that promotes atherosclerosis at normal levels of plasma cholesterol [Metabolism]

October 30th, 2015 by

Approximately one-half of patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins (α-defs) on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition and atherogenesis using transgenic mice expressing human α-defs in their polymorphonuclear leukocytes (Def+/+). Def+/+ mice develop α-def/LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def+/+ to WT mice increased LDL clearance, increased vascular permeability and increased vascular deposition of LDL, while transplantation of WT bone marrow to Def+/+ mice prevented these outcomes. The same outcome was obtained by treating Def+/+ mice with colchicine to inhibit release of α-defs. These studies identify a potential new link between inflammation and the development of atherosclerosis.
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Metabolic cooperation of glucose and glutamine is essential for the lytic cycle of obligate intracellular parasite Toxoplasma gondii [Metabolism]

October 30th, 2015 by Nitzsche, R., Zagoriy, V., Lucius, R., Gupta, N.

Toxoplasma gondii is a widespread protozoan parasite, infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here, we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the TCA cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. Growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine, but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.
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