The overall goal of this proposal is to characterize specific signal transduction mechanisms that are activated after neutrophil stimulation and are involved in heart injury. Neutrophils release lethal oxygen radicals (such as superoxide anions) that are capable of exacerbating tissue damage, as encountered after the infiltration of these cells during post-ischemic myocardial necrosis and reperfusion injury. The enzyme phospholipase D (PLD) is central to the production of PA, a putative second messenger involved in the release of superoxide anions by neutrophils. The authors found that a PLD activity can be specifically immunoprecipitated in its active form with anti-phospho-tyrosine antibodies from cell lysates. The molecular weight of neutrophil PLD as well as the intracellular localization are different from those reported for other mammalian cells. Additionally, the PLD activity is found increased in adherent neutrophils in conditions in which superoxide release is activated. As a result, it is now hypothesized that a novel isoform of PLD in human neutrophils is regulated through tyrosine phosphorylation, which allows the enzyme to become active and synthesize PA, that in turn triggers the release of oxygen radicals.
The Specific Aims of this proposal are: (1) Purify and characterize a novel granulocyte PLD isoform by column chromatography and by molecular biology techniques, using the sequenced purified protein (PY-PLD) as well as the existing mammalian PLD cDNA sequence (PLD1). (2) Determine the mechanism of granulocyte PLD regulation, particularly the phosphorylating kinase, the site(s) of phosphorylation and the role of small GTP-binding proteins, using immunoprecipitation with antibodies against epitope-tagged proteins and in vitro enzymatic assays. (3) Elucidate the role of PY-PLD in the NADPH oxidase system, by blocking superoxide release with PLD antibodies in vitro and in vivo. These studies will result in a clearer understanding of the oxidative processes involved in heart injury and suggest novel therapeutic interventions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL056653-02
Application #
2901252
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Project Start
1998-04-01
Project End
2003-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Wright State University
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Dayton
State
OH
Country
United States
Zip Code
45435
Gomez-Cambronero, Julian; Fite, Kristen; Miller, Taylor E (2018) How miRs and mRNA deadenylases could post-transcriptionally regulate expression of tumor-promoting protein PLD. Adv Biol Regul 68:107-119
Ganesan, Ramya; Henkels, Karen M; Wrenshall, Lucile E et al. (2018) Oxidized LDL phagocytosis during foam cell formation in atherosclerotic plaques relies on a PLD2-CD36 functional interdependence. J Leukoc Biol 103:867-883
Miller, Taylor E; Gomez-Cambronero, Julian (2017) A feedback mechanism between PLD and deadenylase PARN for the shortening of eukaryotic poly(A) mRNA tails that is deregulated in cancer cells. Biol Open 6:176-186
Fite, Kristen; Gomez-Cambronero, Julian (2016) Down-regulation of MicroRNAs (MiRs) 203, 887, 3619 and 182 Prevents Vimentin-triggered, Phospholipase D (PLD)-mediated Cancer Cell Invasion. J Biol Chem 291:719-30
Fite, Kristen; Elkhadragy, Lobna; Gomez-Cambronero, Julian (2016) A Repertoire of MicroRNAs Regulates Cancer Cell Starvation by Targeting Phospholipase D in a Feedback Loop That Operates Maximally in Cancer Cells. Mol Cell Biol 36:1078-89
Gomez-Cambronero, Julian (2015) FASEB Science Research Conference on phospholipid cell signaling and metabolism in inflammation and cancer. FASEB J 29:5-10
Mahankali, Madhu; Alter, Gerald; Gomez-Cambronero, Julian (2015) Mechanism of enzymatic reaction and protein-protein interactions of PLD from a 3D structural model. Cell Signal 27:69-81
Henkels, Karen M; Mallets, Elizabeth R; Dennis, Patrick B et al. (2015) S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding. FASEB J 29:1299-313
Mahankali, Madhu; Henkels, Karen M; Speranza, Francis et al. (2015) A non-mitotic role for Aurora kinase A as a direct activator of cell migration upon interaction with PLD, FAK and Src. J Cell Sci 128:516-26
Kotha, Poornima L N; Sharma, Priyanka; Kolawole, Abimbola O et al. (2015) Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response. PLoS Pathog 11:e1004696

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