Publications by authors named "Neil Grimsey"

21 Publications

  • Page 1 of 1

Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function.

Proc Natl Acad Sci U S A 2020 03 18;117(9):5039-5048. Epub 2020 Feb 18.

Department of Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA 92093;

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.
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http://dx.doi.org/10.1073/pnas.1917295117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060683PMC
March 2020

Ubiquitination as a Key Regulator of Endosomal Signaling by GPCRs.

Front Cell Dev Biol 2019 29;7:43. Epub 2019 Mar 29.

Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA, United States.

G protein-coupled receptors (GPCRs) represent the largest family of therapeutic targets for FDA approved drugs. Therefore, understanding the molecular regulation of their signaling pathways is of paramount importance. Similarly, the mitogen activated protein kinase (MAPK) p38 is a critical mediator of proinflammatory disease. Yet despite decades of intense investigation, therapeutically viable inhibitors have struggled to make it into the clinic. New studies describing the regulation and activation of a GPCR dependent atypical p38 signaling pathway represents a novel therapeutic avenue to the treatment of many proinflammatory disorders. These recent studies have defined how thrombin and ADP can induce Src dependent activation of the E3 ubiquitin ligase NEDD4-2. Src dependent phosphorylation of a 2,3-linker peptide releases NEDD4-2 auto-inhibition and triggers the induction of proinflammatory atypical p38 signaling from the endosome. Activation of the atypical p38 pathway requires the direct interaction between an adaptor protein TAB1 and p38, that bypasses the requirement for the classical MKK3/6 dependent activation of p38. Therefore, providing a mechanism to specifically block proinflammatory GPCR atypical p38 activation while leaving basic p38 activity intact. Critically, new studies demonstrated that disruption of the TAB1-p38 interface is a druggable target, that would enable the selective inhibition of proinflammatory p38 signaling and ischemic injury. Atypical p38 signaling is linked to multiple clinically relevant pathologies including inflammation, cardiotoxicity, myocardial ischemia and ischemia reperfusion injury. Therefore, GPCR induced endosomal p38 signaling represents a novel understudied branch of proinflammatory p38 signaling and an ideal potential therapeutic target that warrants further investigation.
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http://dx.doi.org/10.3389/fcell.2019.00043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449645PMC
March 2019

G protein-coupled receptors activate p38 MAPK via a non-canonical TAB1-TAB2- and TAB1-TAB3-dependent pathway in endothelial cells.

J Biol Chem 2019 04 13;294(15):5867-5878. Epub 2019 Feb 13.

From the Department of Pharmacology and

Endothelial dysfunction is induced by inflammatory mediators including multiple G protein-coupled receptor (GPCR) agonists. However, the GPCR signaling pathways that promote endothelial dysfunction are incompletely understood. We previously showed that thrombin promotes endothelial barrier disruption through autophosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via a non-canonical transforming growth factor-β-activated protein kinase-1-binding protein-1 (TAB1) and TAB2-dependent pathway rather than the canonical three-tiered kinase cascade. Here, we sought to determine whether other GPCR agonists stimulate p38 MAPK activation via this non-canonical pathway in human endothelial cells derived from different vascular beds. Using primary human umbilical vein endothelial cells (HUVECs), HUVEC-derived EA.hy926 cells, and human dermal microvascular endothelial cells (HDMECs), we found that both non-canonical and canonical p38 activation pathways components are expressed in these various endothelial cell types, including TAB3, a structurally-related TAB2 homolog. Moreover, multiple GPCRs agonists, including thrombin, histamine, prostaglandin E, and ADP, stimulated robust p38 autophosphorylation, whereas phosphorylation of the upstream MAPKs MAP kinase kinase 3 (MKK3) and MKK6, was virtually undetectable, indicating that non-canonical p38 activation may exist for other GPCRs. Indeed, in EA.hy926 cells, thrombin- and histamine-stimulated p38 activation depended on TAB1-TAB2, whereas in primary HUVECs, both TAB1-TAB2 and TAB1-TAB3 were required for p38 activation. In HDMECs, thrombin-induced p38 activation depended on TAB1-TAB3, but histamine-induced p38 activation required TAB1-TAB2. Moreover, thrombin- and histamine-stimulated interleukin-6 production required both TAB1-TAB2 and TAB1-TAB3 in HUVEC. We conclude that multiple GPCR agonists utilize non-canonical TAB1-TAB2 and TAB1-TAB3-dependent p38 activation to promote endothelial inflammatory responses.
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http://dx.doi.org/10.1074/jbc.RA119.007495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463731PMC
April 2019

A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling.

Cell Rep 2018 09;24(12):3312-3323.e5

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Ubiquitination is essential for protein degradation and signaling and pivotal to many physiological processes. Ubiquitination of a subset of G-protein-coupled receptors (GPCRs) by the E3 ligase NEDD4-2 is required for p38 activation, but how GPCRs activate NEDD4-2 to promote ubiquitin-mediated signaling is not known. Here, we report that the GPCR protease-activated receptor-1 (PAR1) stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4-2 to promote p38 signaling and endothelial barrier disruption. Using mass spectrometry, we identified a unique phosphorylated tyrosine (Y)-485 within the 2,3-linker peptide between WW domain 2 and 3 of NEDD4-2 in agonist-stimulated cells. Mutation of NEDD4-2 Y485 impaired E3 ligase activity and failed to rescue PAR1-stimulated p38 activation and endothelial barrier permeability. The purinergic P2Y receptor also required c-Src and NEDD4-2 tyrosine phosphorylation for p38 activation. These studies reveal a novel role for c-Src in GPCR-induced NEDD4-2 activation, which is critical for driving ubiquitin-mediated p38 inflammatory signaling.
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http://dx.doi.org/10.1016/j.celrep.2018.08.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226018PMC
September 2018

The unfolded protein response regulator ATF6 promotes mesodermal differentiation.

Sci Signal 2018 02 13;11(517). Epub 2018 Feb 13.

Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA.

encodes a transcription factor that is anchored in the endoplasmic reticulum (ER) and activated during the unfolded protein response (UPR) to protect cells from ER stress. Deletion of the isoform activating transcription factor 6α (ATF6α) and its paralog ATF6β results in embryonic lethality and notochord dysgenesis in nonhuman vertebrates, and loss-of-function mutations in ATF6α are associated with malformed neuroretina and congenital vision loss in humans. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated this hypothesis using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a small-molecule ATF6 agonist and, conversely, inhibited ATF6 using induced pluripotent stem cells from patients with mutations. We found that ATF6 suppressed pluripotency, enhanced differentiation, and unexpectedly directed mesodermal cell fate. Our findings reveal a role for ATF6 during differentiation and identify a new strategy to generate mesodermal tissues through the modulation of the ATF6 arm of the UPR.
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http://dx.doi.org/10.1126/scisignal.aan5785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957084PMC
February 2018

ALIX Regulates the Ubiquitin-Independent Lysosomal Sorting of the P2Y1 Purinergic Receptor via a YPX3L Motif.

PLoS One 2016 14;11(6):e0157587. Epub 2016 Jun 14.

Department of Pharmacology, School of Medicine, University of California San Diego, La Jolla, CA 92093, United States of America.

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination. Here, we describe an ubiquitin-independent lysosomal sorting pathway for the purinergic GPCR P2Y1. After activation, P2Y1 sorts to lysosomes for degradation independent of direct ubiquitination that is mediated by a YPX3L motif within the second intracellular loop that serves as a binding site for the adaptor protein ALIX. Depletion of ALIX or site-directed mutation of the YPX3L motif inhibits P2Y1 sorting into the lumen of multivesicular endosomes/lysosomes and degradation. These findings confirm the function of YPX3L motifs as lysosomal targeting sequences for GPCRs and demonstrate that ALIX mediates the ubiquitin-independent degradation of certain GPCRs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157587PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907476PMC
July 2017

Integration of endothelial protease-activated receptor-1 inflammatory signaling by ubiquitin.

Curr Opin Hematol 2016 May;23(3):274-9

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California, USA.

Purpose Of Review: The maintenance and integrity of the endothelial barrier is essential for vascular homeostasis. Endothelial barrier dysfunction is mediated by various inflammatory factors, many of which act through G protein-coupled receptors including protease-activated receptors (PARs). PARs are expressed in multiple cell types in the vasculature and mediate cellular responses to thrombin, the key effector protease of the coagulation cascade. Thrombin activation of PAR1 induces endothelial barrier permeability through multiple pathways. Here, we discuss the mechanism by which thrombin activation of PAR1 promotes endothelial barrier breakdown and highlight recent advances that have provided new insight into molecular mechanisms that control endothelial barrier integrity.

Recent Findings: Although the signal transduction pathways induced by thrombin activation of PAR1 in endothelial cells have been extensively studied, the key regulatory mechanisms remain poorly understood. Posttranslational modifications are integral to the regulation of PAR1 signaling and recent studies suggest a novel function for ubiquitination of PAR1 in regulation of endothelial barrier permeability.

Summary: An understanding of how endothelial barrier permeability is regulated by thrombin activation of PAR1 is important for the discovery of new drug targets that can be manipulated to control endothelial barrier permeability and prevent progression of vascular inflammation.
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http://dx.doi.org/10.1097/MOH.0000000000000232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978167PMC
May 2016

Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins.

J Biol Chem 2016 Jan 3;291(5):2223-36. Epub 2015 Dec 3.

From the Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms.
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http://dx.doi.org/10.1074/jbc.M115.702993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732206PMC
January 2016

Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes.

J Cell Biol 2015 Sep 21;210(7):1117-31. Epub 2015 Sep 21.

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, 92093

Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β-activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2-mediated ubiquitination and TAB1-TAB2. TAB1-TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.
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http://dx.doi.org/10.1083/jcb.201504007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586747PMC
September 2015

A general method for site specific fluorescent labeling of recombinant chemokines.

PLoS One 2014 28;9(1):e81454. Epub 2014 Jan 28.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11-12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4 °C, and co-internalization with their receptors at 37 °C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081454PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3904831PMC
December 2014

Endosomal signaling by protease-activated receptors.

Methods Enzymol 2014 ;535:389-401

Department of Pharmacology, School of Medicine, University of California, La Jolla, California, USA. Electronic address:

Protease-activated receptors (PARs) are a family of G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. There are four members of the PAR family including: PAR1, PAR2, PAR3, and PAR4. PARs are expressed primarily in the cells of the vasculature and elicit cellular responses to coagulant and anticoagulant proteases. PAR1 exemplifies the unusual proteolytic mechanism of receptor activation. Thrombin binds to and cleaves the N-terminal exodomain of PAR1, generating a new N-terminus that functions as a tethered ligand. The N-terminal tethered ligand domain of PAR1 binds intramolecularly to the receptor to trigger transmembrane signaling and cannot diffuse away. Similar to other GPCRs, activation of PARs promotes coupling to heterotrimeric G proteins at the plasma membrane. After activation, PARs are rapidly internalized to endosomes and then sorted to lysosomes and degraded. Internalization functions to uncouple PARs from heterotrimeric G proteins at the cell surface. However, recent studies indicate that activated internalized PARs signal from endosomes through the recruitment of β-arrestins and potentially other pathways. Here, we provide an overview of methods and strategies used to examine endosomal signaling by PARs.
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http://dx.doi.org/10.1016/B978-0-12-397925-4.00022-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085098PMC
August 2014

Distinct roles of the phosphatidate phosphatases lipin 1 and 2 during adipogenesis and lipid droplet biogenesis in 3T3-L1 cells.

J Biol Chem 2013 Nov 16;288(48):34502-13. Epub 2013 Oct 16.

From the Cambridge Institute for Medical Research, University of Cambridge, CB2 0XY Cambridge, United Kingdom.

Lipins are evolutionarily conserved Mg(2+)-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.
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http://dx.doi.org/10.1074/jbc.M113.488445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843065PMC
November 2013

Adaptor protein complex-2 (AP-2) and epsin-1 mediate protease-activated receptor-1 internalization via phosphorylation- and ubiquitination-dependent sorting signals.

J Biol Chem 2011 Nov 30;286(47):40760-70. Epub 2011 Sep 30.

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.
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http://dx.doi.org/10.1074/jbc.M111.299776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220480PMC
November 2011

Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection.

J Clin Invest 2011 Sep 1;121(9):3554-63. Epub 2011 Aug 1.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.

Azithromycin is a potent macrolide antibiotic with poorly understood antiinflammatory properties. Long-term use of azithromycin in patients with chronic inflammatory lung diseases, such as cystic fibrosis (CF), results in improved outcomes. Paradoxically, a recent study reported that azithromycin use in patients with CF is associated with increased infection with nontuberculous mycobacteria (NTM). Here, we confirm that long-term azithromycin use by adults with CF is associated with the development of infection with NTM, particularly the multi-drug-resistant species Mycobacterium abscessus, and identify an underlying mechanism. We found that in primary human macrophages, concentrations of azithromycin achieved during therapeutic dosing blocked autophagosome clearance by preventing lysosomal acidification, thereby impairing autophagic and phagosomal degradation. As a consequence, azithromycin treatment inhibited intracellular killing of mycobacteria within macrophages and resulted in chronic infection with NTM in mice. Our findings emphasize the essential role for autophagy in the host response to infection with NTM, reveal why chronic use of azithromycin may predispose to mycobacterial disease, and highlight the dangers of inadvertent pharmacological blockade of autophagy in patients at risk of infection with drug-resistant pathogens.
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http://dx.doi.org/10.1172/JCI46095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163956PMC
September 2011

Regulation of protease-activated receptor signaling by post-translational modifications.

IUBMB Life 2011 Jun 24;63(6):403-11. Epub 2011 Mar 24.

Department of Pharmacology, School of Medicine, University of California, San Diego, CA 92093-0636, USA.

Protease-activated receptors (PARs) are a unique family of G-protein-coupled receptors (GPCRs) that are irreversibly activated following proteolytic cleavage of their extracellular N-terminus. PARs play critical functions in hemostasis, thrombosis, inflammation, embryonic development, and cancer progression. Because of the irreversible proteolytic nature of PAR activation, signaling by the receptors is tightly regulated. Three distinct processes including desensitization, internalization, and lysosomal degradation, regulate the temporal and spatial aspects of activated PAR signaling. Post-translational modifications play a critical role in regulating each of these processes and here we review the nature of PAR post-translational modifications and their importance in signal regulation. The PARs are activated by numerous proteases, and some can elicit distinct cellular responses, how this biased agonism is determined is unknown. Further study of the function of post-translational modifications of the PARs will lead to a greater understanding of the physiological regulation of baised agonism and how PAR signaling is precisely controlled in different cellular contexts.
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http://dx.doi.org/10.1002/iub.442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121918PMC
June 2011

Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2.

J Biol Chem 2008 Oct 11;283(43):29166-74. Epub 2008 Aug 11.

Cambridge Institute for Medical Research, University of Cambridge, Hills Road, CB2 0XY Cambridge, United Kingdom.

Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.
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http://dx.doi.org/10.1074/jbc.M804278200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570901PMC
October 2008

Evaluating the role of LPIN1 variation in insulin resistance, body weight, and human lipodystrophy in U.K. Populations.

Diabetes 2008 Sep 30;57(9):2527-33. Epub 2008 Jun 30.

Metabolic Disease Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, U.K.

Objective: Loss of lipin 1 activity causes lipodystrophy and insulin resistance in the fld mouse, and LPIN1 expression and common genetic variation were recently suggested to influence adiposity and insulin sensitivity in humans. We aimed to conduct a comprehensive association study to clarify the influence of common LPIN1 variation on adiposity and insulin sensitivity in U.K. populations and to examine the role of LPIN1 mutations in insulin resistance syndromes.

Research Design And Method: Twenty-two single nucleotide polymorphisms tagging common LPIN1 variation were genotyped in Medical Research Council (MRC) Ely (n = 1,709) and Hertfordshire (n = 2,901) population-based cohorts. LPIN1 exons, exon/intron boundaries, and 3' untranslated region were sequenced in 158 patients with idiopathic severe insulin resistance (including 23 lipodystrophic patients) and 48 control subjects.

Results: We found no association between LPIN1 single nucleotide polymorphisms and fasting insulin but report a nominal association between rs13412852 and BMI (P = 0.042) in a meta-analysis of 8,504 samples from in-house and publicly available studies. Three rare nonsynonymous variants (A353T, R552K, and G582R) were detected in severely insulin-resistant patients. However, these did not cosegregate with disease in affected families, and Lipin1 protein expression and phosphorylation in patients with variants were indistinguishable from those in control subjects.

Conclusions: Our data do not support a major effect of common LPIN1 variation on metabolic traits and suggest that mutations in LPIN1 are not a common cause of lipodystrophy in humans. The nominal associations with BMI and other metabolic traits in U.K. cohorts require replication in larger cohorts.
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http://dx.doi.org/10.2337/db08-0422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2518506PMC
September 2008

The human lipodystrophy gene BSCL2/seipin may be essential for normal adipocyte differentiation.

Diabetes 2008 Aug 5;57(8):2055-60. Epub 2008 May 5.

University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK.

Objective: Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) is a recessive disorder featuring near complete absence of adipose tissue. Remarkably, although the causative gene, BSCL2, has been known for several years, its molecular function and its role in adipose tissue development have not been elucidated. Therefore, we examined whether BSCL2 is involved in the regulation of adipocyte differentiation and the mechanism whereby pathogenic mutations in BSCL2 cause lipodystrophy.

Research Design And Methods: Following the characterization of BSCL2 expression in developing adipocytes, C3H10T1/2 mesenchymal stem cells were generated in which BSCL2 expression was knocked down using short hairpin RNA (shRNA). These cells were used to investigate whether BSCL2 is required for adipogenesis. BSCL2 constructs harboring pathogenic mutations known to cause lipodystrophy were also generated and characterized.

Results: BSCL2 expression was strongly induced during adipocyte differentiation, and the induction of BSCL2 expression was essential for adipogenesis to occur. The initial induction of key adipogenic transcription factors, including peroxisome proliferator-activated receptor (PPAR)gamma and CAAT/enhancer-binding protein-alpha, was preserved in cells lacking BSCL2. However, the expression of these critical factors was not sustained, suggesting that the activity of PPARgamma was impaired. Moreover, expression of key genes mediating triglyceride synthesis, including AGPAT2, lipin 1, and DGAT2, was persistently reduced and lipid accumulation was inhibited. Analysis of pathogenic missense mutants of BSCL2 revealed that the amino acid substitution A212P causes aberrant targeting of BSCL2 within the cell, suggesting that subcellular localization of BSCL2 may be critical to its function.

Conclusions: This study demonstrates that BSCL2 is an essential, cell-autonomous regulator of adipogenesis.
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http://dx.doi.org/10.2337/db08-0184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2494687PMC
August 2008

Two human ARFGAPs associated with COP-I-coated vesicles.

Traffic 2007 Nov 29;8(11):1644-55. Epub 2007 Sep 29.

Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 2XY, United Kingdom.

ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.
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http://dx.doi.org/10.1111/j.1600-0854.2007.00631.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171037PMC
November 2007

Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.

J Biol Chem 2006 Nov 12;281(45):34537-48. Epub 2006 Sep 12.

Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, CB2 2XY Cambridge, United Kingdom.

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1delta cells.
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http://dx.doi.org/10.1074/jbc.M606654200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1769310PMC
November 2006

The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.

EMBO J 2005 Jun 5;24(11):1931-41. Epub 2005 May 5.

WellcomeTrust/Cancer Research UK Gurdon Institute, Cambridge, UK.

Remodelling of the nuclear membrane is essential for the dynamic changes of nuclear architecture at different stages of the cell cycle and during cell differentiation. The molecular mechanism underlying the regulation of nuclear membrane biogenesis is not known. Here we show that Smp2, the yeast homologue of mammalian lipin, is a key regulator of nuclear membrane growth during the cell cycle. Smp2 is phosphorylated by Cdc28/Cdk1 and dephosphorylated by a nuclear/endoplasmic reticulum (ER) membrane-localized CPD phosphatase complex consisting of Nem1 and Spo7. Loss of either SMP2 or its dephosphorylated form causes transcriptional upregulation of key enzymes involved in lipid biosynthesis concurrent with a massive expansion of the nucleus. Conversely, constitutive dephosphorylation of Smp2 inhibits cell division. We show that Smp2 associates with the promoters of phospholipid biosynthetic enzymes in a Nem1-Spo7-dependent manner. Our data suggest that Smp2 is a critical factor in coordinating phospholipid biosynthesis at the nuclear/ER membrane with nuclear growth during the cell cycle.
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http://dx.doi.org/10.1038/sj.emboj.7600672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142606PMC
June 2005