24 results on '"Stow, Jennifer L."'
Search Results
2. Macropinocytosis: Insights from immunology and cancer.
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Stow, Jennifer L., Hung, Yu, and Wall, Adam A.
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FLUID control , *GALECTINS , *BIOPHYSICS , *IMMUNOLOGY , *METABOLIC regulation - Abstract
Macropinocytosis is increasingly recognized for its versatile adaptations and functions as a highly conserved, ubiquitous pathway for the bulk uptake of fluid, particulate cargo, and membranes. Innate immune cells and transformed cancer cells share the capacity for both constitutive and induced macropinocytosis, which is used for immune surveillance, ingestion of pathogens, immune response shaping, and enhancement of scavenging for nutrients as fuel for cell survival and proliferation. Immunology and cancer biology are leading a resurgence of interest in defining the molecular and physiological regulation of macropinocytosis, partly in pursuit of ways to control macropinocytic uptake in disease settings. New approaches, including high-resolution live imaging, screening of cell surface molecular inventories, biophysics, and exploration of cell microenvironments, have converged to provide new insights into macropinosome induction, formation, and maturation. Recent studies reveal mechanisms for fluid control in and by macrophage macropinosomes that impinge on membrane trafficking and cell migration. EGFR, PTEN, V-ATPase, syndecan 1, and galectin-3 have roles variably in the metabolic regulation of Ras or PI3K signaling for Rac1-mediated macropinocytosis in cancer. These molecular pathways and mechanisms contribute to the impressive adaptability of macropinocytosis in many cells and tissues and in disease. [ABSTRACT FROM AUTHOR]
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- 2020
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3. TLR Crosstalk Activates LRP1 to Recruit Rab8a and PI3Kγ for Suppression of Inflammatory Responses.
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Luo, Lin, Wall, Adam A., Tong, Samuel J., Hung, Yu, Xiao, Zhijian, Tarique, Abdullah A., Sly, Peter D., Fantino, Emmanuelle, Marzolo, María-Paz, and Stow, Jennifer L.
- Abstract
Summary The multi-ligand endocytic receptor, low-density lipoprotein-receptor-related protein 1 (LRP1), has anti-inflammatory roles in disease. Here, we reveal that pathogen-activated Toll-like receptors (TLRs) activate LRP1 in human and mouse primary macrophages, resulting in phosphorylation of LRP1 at Y4507. In turn, this allows LRP1 to activate and recruit the guanosine triphosphatase (GTPase), Rab8a, with p110γ/p101 as its phosphatidylinositol 3-kinase (PI3K) effector complex. PI3Kγ is a known regulator of TLR signaling and macrophage reprogramming. LRP1 coincides with Rab8a at signaling sites on macropinosomal membranes. In LRP1-deficient cells, TLR-induced Rab8 activation is abolished. CRISPR-mediated knockout of LRP1 in macrophages alters Akt/mTOR signaling and produces a pro-inflammatory bias in cytokine outputs, mimicking the Rab8a knockout and PI3Kγ-null phenotype. Thus, TLR-LRP1 crosstalk activates the Rab8a/PI3Kγ complex for reprogramming macrophages, revealing this as a key mechanism through which LRP1 helps to suppress inflammation. Graphical Abstract Highlights • Multiple TLRs activate LRP1, resulting in phosphorylation of LRP1 • TLR-induced phosphorylation of LRP1 recruits Rab8a with its PI3K effector p110γ/p101 • LRP1/Rab8a/ PI3Kγ activates Akt/mTOR signaling and biases cytokine outputs • The LRP1/Rab8a/PI3Kγ complex reprograms macrophages and restrains inflammation Luo et al. show that the multifunctional endocytic receptor, LRP1, is crosstalk activated by agonist-activated TLRs on macrophages. Phosphorylated LRP1 recruits a Rab/PI3K complex that activates Akt/mTOR signaling to repolarize macrophages and bias inflammatory cytokine outputs. Through this mechanism, LRP1 helps to suppress TLR-induced inflammation. [ABSTRACT FROM AUTHOR]
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- 2018
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4. Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion.
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Monteleone, Mercedes, Stanley, Amanda C., Chen, Kaiwen W., Brown, Darren L., Bezbradica, Jelena S., von Pein, Jessica B., Holley, Caroline L., Boucher, Dave, Shakespear, Melanie R., Kapetanovic, Ronan, Rolfes, Verena, Sweet, Matthew J., Stow, Jennifer L., and Schroder, Kate
- Abstract
Summary IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion. [ABSTRACT FROM AUTHOR]
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- 2018
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5. Host glycocalyx captures HIV proximal to the cell surface via oligomannose-GlcNAc glycan-glycan interactions to support viral entry.
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Spillings, Belinda L., Day, Christopher J., Garcia-Minambres, Albert, Aggarwal, Anupriya, Condon, Nicholas D., Haselhorst, Thomas, Purcell, Damian F.J., Turville, Stuart G., Stow, Jennifer L., Jennings, Michael P., and Mak, Johnson
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Here, we present ultrastructural analyses showing that incoming HIV are captured near the lymphocyte surface in a virion-glycan-dependent manner. Biophysical analyses show that removal of either virion- or cell-associated N-glycans impairs virus-cell binding, and a similar glycan-dependent relationship is observed between purified HIV envelope (Env) and primary T cells. Trimming of N-glycans from either HIV or Env does not inhibit protein-protein interactions. Glycan arrays reveal HIV preferentially binds to N-acetylglucosamine and mannose. Interfering with these glycan-based interactions reduces HIV infectivity. These glycan interactions are distinct from previously reported glycan-lectin and non-specific electrostatic charge-based interactions. Specific glycan-glycan-mediated attachment occurs prior to virus entry and enhances efficiency of infection. Binding and fluorescent imaging data support glycan-glycan interactions as being responsible, at least in part, for initiating contact between HIV and the host cell, prior to viral Env-cellular CD4 engagement. [Display omitted] • Viral glycan shield acts as a cellular attachment factor • Viral oligomannose initiates host cell attachment • This initial attachment is a specific, non-electrostatic interaction • Blocking this interaction may present a pan-specific suppression mechanism Contrasting with sugar-protein interactions, Spillings et al. describe a specific, non-electrostatic sugar-sugar interaction facilitating HIV and host cell attachment, which involves oligomannose, previously thought to function as a shield. Many human viruses possess oligomannose-enriched glycan shields, suggesting that sugar-mediated interactions may be a general principle for initial viral attachment [ABSTRACT FROM AUTHOR]
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- 2022
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6. Intracellular trafficking and secretion of inflammatory cytokines.
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Stow, Jennifer L. and Murray, Rachael Z.
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INFLAMMATION , *CYTOKINES , *CELLULAR signal transduction , *PROTEIN synthesis , *MESSENGER RNA , *CELL membranes - Abstract
Abstract: The secretion of cytokines by immune cells plays a significant role in determining the course of an inflammatory response. The levels and timing of each cytokine released are critical for mounting an effective but confined response, whereas excessive or dysregulated inflammation contributes to many diseases. Cytokines are both culprits and targets for effective treatments in some diseases. The multiple points and mechanisms that have evolved for cellular control of cytokine secretion highlight the potency of these mediators and the fine tuning required to manage inflammation. Cytokine production in cells is regulated by cell signaling, and at mRNA and protein synthesis levels. Thereafter, the intracellular transport pathways and molecular trafficking machinery have intricate and essential roles in dictating the release and activity of cytokines. The trafficking machinery and secretory (exocytic) pathways are complex and highly regulated in many cells, involving specialized membranes, molecules and organelles that enable these cells to deliver cytokines to often-distinct areas of the cell surface, in a timely manner. This review provides an overview of secretory pathways – both conventional and unconventional – and key families of trafficking machinery. The prevailing knowledge about the trafficking and secretion of a number of individual cytokines is also summarized. In conclusion, we present emerging concepts about the functional plasticity of secretory pathways and their modulation for controlling cytokines and inflammation. [Copyright &y& Elsevier]
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- 2013
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7. Cytokine secretion in macrophages and other cells: Pathways and mediators
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Stow, Jennifer L., Ching Low, Pei, Offenhäuser, Carolin, and Sangermani, Daniele
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CYTOKINES , *GREEN fluorescent protein , *CELLULAR immunity , *GROWTH factors - Abstract
Abstract: Cytokines and other immune mediators are secreted by cells of the immune system during immune responses and as a means of communication. While the functions of these cytokines, chemokines and mediators are well known, the intracellular pathways that lead to their secretion by different cells are only now being fully documented. Cytokines in some cells are released from secretory granules while in other cells they are released via constitutive secretory pathways that instead have more dynamic vesicular carriers. Recent studies have revealed that newly synthesized cytokines can be routed via compartments such as recycling endosomes prior to their secretion. Here we describe and show examples of some of the pathways used for cytokine trafficking and release in macrophages, including some of the cellular machinery required for this transport. Increasingly, these trafficking pathways are revealed as having important regulatory roles in the execution of immune responses. [Copyright &y& Elsevier]
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- 2009
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8. The ins and outs of E-cadherin trafficking
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Bryant, David M. and Stow, Jennifer L.
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PROTEINS , *CELL adhesion , *TUMOR suppressor genes , *TUMOR suppressor proteins , *CANCER - Abstract
One way of controlling the activity of E-cadherin – a protein that is, simultaneously, a major cell-adhesion molecule, a powerful tumour suppressor, a determinant of cell polarity and a partner to the potent catenin signalling molecules – is to keep it on the move. During the past two decades, many insights into the fundamental role of E-cadherin in these processes have been garnered. Studies during the past five years have begun to reveal the importance of intracellular trafficking as a means of regulating the functions of E-cadherin. E-cadherin is trafficked to and from the cell surface by exocytic and multiple endocytic pathways. In this article, we survey the vesicle-trafficking machinery that is responsible for the sorting, transport, actin association and vesicle targeting of E-cadherin to regulate its movement and function during growth and development and, possibly, in cancer. [Copyright &y& Elsevier]
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- 2004
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9. SCIMP is a spatiotemporal transmembrane scaffold for Erk1/2 to direct pro-inflammatory signaling in TLR-activated macrophages.
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Lucas, Richard M., Liu, Liping, Curson, James E.B., Koh, Yvette W.H., Tuladhar, Neeraj, Condon, Nicholas D., Das Gupta, Kaustav, Burgener, Sabrina S., Schroder, Kate, Ingley, Evan, Sweet, Matthew J., Stow, Jennifer L., and Luo, Lin
- Abstract
Immune cells are armed with Toll-like receptors (TLRs) for sensing and responding to pathogens and other danger cues. The role of extracellular-signal-regulated kinases 1/2 (Erk1/2) in TLR signaling remains enigmatic, with both pro- and anti-inflammatory functions described. We reveal here that the immune-specific transmembrane adaptor SCIMP is a direct scaffold for Erk1/2 in TLR pathways, with high-resolution, live-cell imaging revealing that SCIMP guides the spatial and temporal recruitment of Erk2 to membrane ruffles and macropinosomes for pro-inflammatory TLR4 signaling. SCIMP-deficient mice display defects in Erk1/2 recruitment to TLR4, c-Fos activation, and pro-inflammatory cytokine production, with these effects being phenocopied by Erk1/2 signaling inhibition. Our findings thus delineate a selective role for SCIMP as a key scaffold for the membrane recruitment of Erk1/2 kinase to initiate TLR-mediated pro-inflammatory responses in macrophages. [Display omitted] • Immune-specific TLR adaptor SCIMP is a scaffold for MAPK Erk1/2 in macrophages • SCIMP recruits Erk2 to cell surface ruffles and macropinosomes for TLR signaling • SCIMP-scaffolded Erk1/2 activates c-Fos to drive pro-inflammatory cytokine production SCIMP is an immune-specific, transmembrane TLR adaptor genetically associated with inflammatory diseases. In this paper, Lucas et al. show that TLR-activated SCIMP scaffolds the signaling kinase Erk1/2, recruiting it to the cell surface to drive pro-inflammatory cytokine responses in macrophages. Targeting of SCIMP may dampen pro-inflammatory responses in disease settings. [ABSTRACT FROM AUTHOR]
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- 2021
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10. New advances in innate immune endosomal trafficking.
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Stocks, Claudia J., Li, Xichun, and Stow, Jennifer L.
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INFLAMMATORY mediators , *GROWTH factors , *MOLECULAR interactions , *INFLAMMATION , *ORGANELLES , *CHLORIDE channels - Abstract
The exocytic and endocytic intracellular trafficking pathways in innate immune cells are known for mediating the secretion of key inflammatory mediators or the internalization of growth factors, nutrients, antigens, cell debris, pathogens and even therapeutics, respectively. Inside cells, these pathways are intertwined as an elaborate network that supports the regulation of immune functions. Endosomal membranes host dynamic platforms for molecular complexes that control signaling and inflammatory responses. High content screens, coupled with elegant microscopy across the scale of resolving molecular complexes to tracking live cellular organelles, have been employed to generate the studies highlighted here. With a focus on deactivation of STING, scaffolding by SLC15A4/TASL complexes and macropinosome shrinkage via the chloride channel protein TMEM206, new studies are identifying molecules, molecular interactions and mechanisms for immune regulation throughout endosomal pathways. [ABSTRACT FROM AUTHOR]
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- 2024
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11. Endocytosis of Uncleaved Tumor Necrosis Factor-a in Macrophages.
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Shurety, Wenda, Pagan, Julia K., Prins, Johannes B., and Stow, Jennifer L.
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- 2001
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12. Editorial overview: Membrane traffic in the time of COVID-19.
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Brodsky, Frances M. and Stow, Jennifer L.
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ENDOCYTOSIS , *COATED vesicles , *COVID-19 , *ORGANELLE formation , *CELL polarity , *TISSUE differentiation - Published
- 2020
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13. The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs.
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Liping Liu, Lucas, Richard M., Nanson, Jeffrey D., Yan Li, Whitfield, Jason, Curson, James E. B., Tuladhar, Neeraj, Alexandrov, Kirill, Mobli, Mehdi, Sweet, Matthew J., Kobe, Bostjan, Stow, Jennifer L., and Lin Luo
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TOLL-like receptors , *PROTEIN-tyrosine kinases , *SCAFFOLD proteins , *MEMBRANE proteins , *KINASES , *ADAPTOR proteins - Abstract
Innate immune signaling by Toll-like receptors (TLRs) involves receptor phosphorylation, which helps to shape and drive key inflammatory outputs, yet our understanding of the kinases and mechanisms that mediate TLR phosphorylation is incomplete. Spleen tyrosine kinase (Syk) is a nonreceptor protein tyrosine kinase, which is known to relay adaptive and innate immune signaling, including from TLRs. However, TLRs do not contain the conserved dual immunoreceptor tyrosinebased activation motifs that typically recruit Syk to many other receptors. One possibility is that the Syk-TLR association is indirect, relying on an intermediary scaffolding protein. We previously identified a role for the palmitoylated transmembrane adapter protein SCIMP in scaffolding the Src tyrosine kinase Lyn, for TLR phosphorylation, but the role of SCIMP in mediating the interaction between Syk and TLRs has not yet been investigated. Here, we show that SCIMP recruits Syk in response to lipopolysaccharide-mediated TLR4 activation. We also show that Syk contributes to the phosphorylation of SCIMP and TLR4 to enhance their binding. Further evidence pinpoints two specific phosphorylation sites in SCIMP critical for its interaction with Syk-SH2 domains in the absence of immunoreceptor tyrosine-based activation motifs. Finally, using inhibitors and primary macrophages from SCIMP-/- mice, we confirm a functional role for SCIMP-mediated Syk interaction in modulating TLR4 phosphorylation, signaling, and cytokine outputs. In conclusion, we identify SCIMP as a novel, immune-specific Syk scaffold, which can contribute to inflammation through selective TLR-driven inflammatory responses. [ABSTRACT FROM AUTHOR]
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- 2022
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14. Individual Smad2 linker region phosphorylation sites determine the expression of proteoglycan and glycosaminoglycan synthesizing genes.
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Kamato, Danielle, Burch, Micah, Zhou, Ying, Mohamed, Raafat, Stow, Jennifer L., Osman, Narin, Zheng, Wenhua, and Little, Peter J.
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PHOSPHORYLATION , *PROTEOGLYCANS , *TRANSFORMING growth factors , *LIPOPROTEINS , *ATHEROSCLEROSIS - Abstract
Abstract Growth factors such as thrombin and transforming growth factor (TGF)-β facilitate glycosaminoglycan (GAG) chain hyperelongation on proteoglycans, a phenomenon that increases lipoprotein binding in the vessel wall and the development of atherosclerosis. TGF-β signals via canonical carboxy terminal phosphorylation of R-Smads and also non-canonical linker region phosphorylation of R-Smads. The G protein coupled receptor agonist, thrombin, can transactivate the TGF-β receptor leading to both canonical and non-canonical Smad signalling. Linker region phosphorylation drives the expression of genes for the synthesis of the proteoglycan, biglycan. Proteoglycan synthesis involves core protein synthesis, the initiation of GAG chains and the subsequent elongation of GAG chains. We have explored the relationship between the thrombin stimulated phosphorylation of individual serine and threonine sites in the linker region of Smad2 and the expression of GAG initiation xylosyltransferase-1 (XT-1) and GAG elongation chondroitin 4-sulfotransferase-1 (C4ST-1) and chondroitin synthase-1 (CHSY-1) genes. Thrombin stimulated the phosphorylation of all four target residues (Thr220, Ser245, Ser250 and Ser255 residues) with a similar temporal pattern – phosphorylation was maximal at 15 min (the earliest time point studied) and the level of the phospho-proteins declined thereafter over the following 4 h. Jnk, p38 and PI3K, selectively mediated the phosphorylation of the Thr220 residue whereas the serine residues were variously phosphorylated by multiple kinases. Thrombin stimulated the expression of all three genes – XT-1, C4ST-1 and CHSY-1. The three pathways mediating Thr220 phosphorylation were also involved in the expression of XT-1. The target pathways (excluding Jnk) were involved in the expression of the GAG elongation genes (C4ST-1 and CHSY-1). These findings support the contention that individual Smad linker region phosphorylation sites are linked to the expression of genes for the initiation and elongation of GAG chains on proteoglycans. The context of this work is that a specific inhibitor of GAG elongation represents a potential therapeutic agent for preventing GAG elongation and lipid binding and the results indicate that the specificity of the pathways is such that it might be therapeutically feasible to specifically target GAG elongation without interfering with other physiological processes with which proteoglycans are involved. Highlights • Thrombin stimulates Smad linker region phosphorylation. • Thrombin stimulates the expression of glycosaminoglycan synthesizing genes. • Serine residues of Smad linker region correlated with GAG chain elongation. • Threonine residue of Smad linker region correlated with GAG chain initiation. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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15. The Binding of Syndapin SH3 Domain to Dynamin Proline-rich Domain Involves Short and Long Distance Elements.
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Lin Luo, Jing Xue, Ann Kwan, Gamsjaeger, Roland, Wielens, Jerome, von Kleist, Lisa, Cubeddu, Liza, Zhong Guo, Stow, Jennifer L., Parker, Michael W., Mackay, Joel P., and Robinson, Phillip J.
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DYNAMIN (Genetics) , *GUANOSINE triphosphatase , *PROLINE , *PEPTIDES , *C-terminal residues - Abstract
Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786- Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction. [ABSTRACT FROM AUTHOR]
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- 2016
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16. The Inflammasome Adaptor ASC Induces Procaspase-8 Death Effector Domain Filaments.
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Vajjhala, Parimala R., Lu, Alvin, Brown, Darren L., Siew Wai Pang, Sagulenko, Vitaliya, Sester, David P., Cridland, Simon O., Hill, Justine M., Schroder, Kate, Stow, Jennifer L., Hao Wu, and Stacey, Katryn J.
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PROTEIN-protein interactions , *CELL death , *CELLULAR signal transduction , *ENZYME activation , *CASPASES , *ADAPTOR proteins - Abstract
Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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17. Cyclosporin A Decreases Apolipoprotein E Secretion from Human Macrophages via a Protein Phosphatase 2B-dependent and ATP-binding Cassette Transporter A1 (ABCA1 )-independent Pathway.
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Kockx, Maaike, Guo, Dongni Lily, Traini, Mathew, Gau, Katharina, Kay, Jason, Wimmer-Kleikamp, Sabine, Rentero, Carles, Burnett, John R., Le Goff, Wilfried, Van Eck, Miranda, Stow, Jennifer L., Jessup, Wendy, and Kritharides, Leonard
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CYCLOSPORINE , *PHOSPHOPROTEIN phosphatases , *HYPERLIPIDEMIA , *CHOLESTEROL , *ATHEROSCLEROSIS , *INTRACELLULAR pathogens , *BLOOD lipids - Abstract
Cyclosporin A (CsA) is an immunosuppressant that inhibits protein phosphatase 2B (PP2B/calcineurin) and is associated with hyperlipidemia, decreased cholesterol efflux via ATP-binding cassetie transporter Al (ABCA1), and increased risk of atherosclerosis. Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, the secretion of which from human macrophages is regulated by the serine/threonine protein kinase A (PKA) and intracellular calcium (Ca2+) (Koch, M., Guo, D. L., Huby, T., Lesnik, P., Kay, J., Sabaretnam, T., Jary, E., Hill, M., Gaus, K., Chapman, J., Stow, J. L., Jessup, W., and I(ritharides, L. (2007) Circ. Res. 101, 607-616). As PP2B is Ca2+-dependent and has been linked to PKA-dependent processes, we investigated whether CsA modulated apoE secretion. CsA doseand time-dependently inhibited secretion of apoE from primary human macrophages and from Chinese hamster ovary cells stably transfected with human apoE and increased cellular apoE levels without affecting apoE mRNA. [35S)Met kinetic modeling studies showed that CsA inhibited both secretion and degradation of apoE, increasing the half-life of cellular apoE 2-fold. CsA also inhibited secretion from primary human Tangier disease macrophages and from mouse macrophages deficient in ABCA1, indicating that the effect is independent of the known inhibition of ABCA1 by CsA. The role of PP2B in mediating apoE secretion was confirmed using additional peptide and chemical inhibitors of PP2B. Importantly, kinetic modeling, live-cell imaging, and confocal microscopy all indicated that CsA inhibited apoE secretion by mechanisms quite distinct from those of PKA inhibition, most likely inducing accumulation of apoE in the endoplasmic reticulum compartment. Taken together, these results establish a novel mechanism for the pro-atherosclerotic effects of CsA, and establish for the first time a role for PP2B in regulating the intracellular transport and secretion of apoE. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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18. Analysis of Cultured Human Melanocytes Based on Polymorphisms within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P Loci.
- Author
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Cook, Anthony L., Wei Chen, Thurber, Amy E., Smit, Darren J., Smith, Aaron G., Bladen, Timothy G., Brown, Darren L., Duffy, David L., Pastorino, Lorenza, Bianchi-Scarra, Giovanna, Leonard, J. Helen, Stow, Jennifer L., and Sturm, Richard A.
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MELANOCYTES , *NUCLEOTIDES , *GENETIC polymorphisms , *MELANINS , *DERMATOLOGY - Abstract
Single nucleotide polymorphisms (SNPs) within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P genes have been associated with natural variation of pigmentation traits in human populations. Here, we describe the characterization of human primary melanocytic cells genotyped for polymorphisms within the MATP, NCKX5, or OCA2 loci. On the basis of genotype, these cultured cells reflect the phenotypes observed by others in terms of both melanin content and tyrosinase (TYR) activity when comparing skin designated as either “White” or “Black”. We found a statistically significant association of MATP-374L (darker skin) with higher TYR protein abundance that was not observed for any NCKX5-111 or OCA2 rs12913832 allele. MATP-374L/L homozygous strains displayed significantly lower MATP transcript levels compared to MATP-374F/F homozygous cells, but this did not reach statistical significance based on NCKX5 or OCA2 genotype. Similarly, we observed significantly increased levels of OCA2 mRNA in rs12913832-T (brown eye) homozygotes compared to rs12913832-C (blue eye) homozygous strains, which was not observed for MATP or NCKX5 gene transcripts. In genotype–phenotype associations performed on a collection of 226 southern European individuals using these same SNPs, we were able to show strong correlations in MATP-L374F, OCA2, and melanocortin-1 receptor with skin, eye, and hair color variation, respectively.Journal of Investigative Dermatology (2009) 129, 392–405; doi:10.1038/jid.2008.211; published online 24 July 2008 [ABSTRACT FROM AUTHOR]
- Published
- 2009
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19. The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis
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Greenwood, Kathryn P., Daly, Norelle L., Brown, Darren L., Stow, Jennifer L., and Craik, David J.
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PROTEINS , *GENETIC engineering , *CYSTATHIONINE gamma-lyase , *CANCER cells - Abstract
Abstract: The cyclotides are macrocyclic knotted proteins characterized by a compact topology and exceptional stability. Accordingly it has been hypothesized that they may be useful as protein engineering frameworks for the stabilization and delivery of bioactive peptide sequences. This study examined the internalization of cyclotides into mammalian cells, a vital step for the delivery of bioactive peptide sequences to intracellular targets. Although the entry of various linear peptides into cells has been reported previously, this is the first report of internalization of a macrocyclic peptide. Cell uptake was examined for representatives of two cyclotide subfamilies; the first was MCoTI-II, a member of the trypsin inhibitor subfamily, which was internalized by a macrophage and breast cancer cell line and the second, the prototypic cyclotide kalata B1 from the Möbius subfamily, which remained extracellular. Biotin labeled MCoTI-II entered macrophages by macropinocytosis, resulting in vesicular encapsulation without trafficking to lysosomes for degradation. The ready uptake, coupled with low cytotoxicity, indicates that MCoTI-II has the potential to transport grafted bioactivities to intracellular targets, making it a potentially valuable framework in drug design applications. [Copyright &y& Elsevier]
- Published
- 2007
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20. Cytokine Secretion via Cholesterol-rich Lipid Raft-associated SNAREs at the Phagocytic Cup.
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Kay, Jason G., Murray, Rachael Z., Pagan, Julia K., and Stow, Jennifer L.
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MACROPHAGES , *TUMOR necrosis factors , *IMMUNE system , *CELL membranes , *PHAGOCYTOSIS - Abstract
Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor α (TNFα) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFα is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNFα trafficking and secretion. TNFα surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNF~ surface distribution revealed these puncta to be the initial points of TNFα delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response. [ABSTRACT FROM AUTHOR]
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- 2006
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21. Syntaxin 6 and Vti1b Form a Novel SNARE Complex, Which Is Up-regulated in Activated Macrophages to Facilitate Exocytosis of Tumor Necrosis Factor-α.
- Author
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Murray, Rachael Z., Wylie, Fiona G., Khromykh, Tatiana, Hume, David A., and Stow, Jennifer L.
- Subjects
- *
MACROPHAGES , *CYTOKINES , *PROTEINS , *BACTERIA , *CELLS - Abstract
A key function of activated macrophages is to secrete proinflammatory cytokines such as TNFα; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNFα vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNFα trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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22. Contextual Binding of p120[sup ctn] to E-cadherin at the Basolateral Plasma Membrane in Polarized Epithelia.
- Author
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Miranda, Kevin C., Joseph, Shannon R., Yap, Alpha S., Teasdale, Rohan D., and Stow, Jennifer L.
- Subjects
- *
CELL communication , *EPITHELIAL cells , *CELLS - Abstract
E-cadherin-catenin complexes mediate cell-cell adhesion on the basolateral membrane of epithelial cells. The cytoplasmic tail of E-cadherin supports multiple protein interactions, including binding of β-catenin at the C terminus and of p120[sup ctn] to the juxtamembrane domain. The temporal assembly and polarized trafficking of the complex or its individual components to the basolateral membrane are not fully understood. In Madin-Darby canine kidney cells at steady state and after treatment with cycloheximide or temperature blocks, E-cadherin and β-catenin localized to the Golgi complex, but p120[sup ctn] was found only at the basolateral plasma membrane. We previously identified a dileucine sorting motif (Leu[sup 586]Leu[sup 587], termed S1) in the juxtamembrane domain of Ecadherin and now show that it is required to target full-length E-cadherin to the basolateral membrane. Removal of SI resulted in missorting of E-cadherin mutants (EcadΔS1) to the apical membrane; β-catenin was simultaneously missorted and appeared at the apical membrane, p120[sup ctn] was not mistargeted with EcadΔS1, but could be recruited to the E-cadherin-catenin complex only at the basolateral membrane. These findings help define the temporal assembly and sorting of the E-cadherin-catenin complex and show that membrane recruitment of p120[sup ctn] in polarized cells is contextual and confined to the basolateral membrane. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
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23. Characterization of E-cadherin Endocytosis in Isolated MCF-7 and Chinese Hamster Ovary Cells.
- Author
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Paterson, Andrew D., Parton, Robert G., Ferguson, Charles, Stow, Jennifer L., and Yap, Alpha S.
- Subjects
- *
CELL adhesion molecules , *ENDOCYTOSIS - Abstract
Focuses on the characterization of E-cadherin endocytosis in isolated MCF-7 and Chinese hamster ovary cells. Comparison of E-cadherin and clathrin-mediated uptake pathways; Characterization of E-cadherin internalization by immunoelectron microscopy.
- Published
- 2003
- Full Text
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24. GRIP Domain-mediated Targeting of Two New Coiled-coil Proteins, GCC88 and GCC185, to Subcompartments of the trans-Golgi Network.
- Author
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Luke, Michael R., Kjer-Nielsen, Lars, Brown, Darren L., Stow, Jennifer L., and Gleeson, Paul A.
- Subjects
- *
GOLGI apparatus , *PROTEINS , *BIOLOGICAL transport - Abstract
Focuses on the GRIP domain-mediated targeting of two coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network (TGN). Localization of the proteins in HeLa cells of TGN; Perturbation of a domain of the TGN associated with the membrane transport of TGN38 with overexpression of GCC88.
- Published
- 2003
- Full Text
- View/download PDF
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