Radiation Protection: Phospholipase C,LAMP and

Phospholipase C, LAMP inhibition.Dmitri Popov, PhD, Radiobiology. Canada.

MD (Russia).

Phospholipase C, LAMP….• Research Proposal:

Radiation Protection: Phospholipase C, LAMP and Phospholipase C, LAMP inhibition.• Dmitri Popov• Full-text · Research Proposal · Apr 2016• Add resources• File name: RadProtectionPhospholipaseC.pptx

DOI: 10.13140/RG.2.1.2121.7680

Phospholipase C.

• Phospholipase C (PLC) is a class of membrane-associated enzymes that cleave phospholipids just before the phosphate group (see figure). It is most commonly taken to be synonymous with the human forms of this enzyme, which play an important role in eukaryotic cell physiology, in particular signal transductionpathways. There are thirteen kinds of mammalian phospholipase C that are classified into six isotypes (β, γ, δ, ε, ζ, η) according to structure. Each PLC has unique and overlapping controls over expression and subcellular distribution. Activators of each PLC vary, but typically include heterotrimeric G protein sub-units, protein tyrosine kinases, small G proteins, Ca2+, and phospholipids. https://en.wikipedia.org/wiki/Phospholipase_C

Phospholipase C.• A LARGE NUMBER of extracellular signals stimulate hydrolysis of

phosphatidylinositol 4,5-bisphophate (PIP2) by phosphoinositide-specific phospholipase C (PI-PLC) . • For the regulation of cellular processes, the best documented

consequence of this hydrolysis is the generation of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, involved in calcium release from intracellular stores and stimulation of protein kinase C isozymes.• Phosphoinositide-specific phospholipase C: structural basis for catalysis

and regulatory interactions Matilda Katan and Roger L. Williams. Seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 8, 1997: pp 287–296

Phospholipase C.• The primary catalyzed reaction of PLC occurs on an insoluble substrate at a

lipid-water interface. The residues in the active site are conserved in all PLC isotypes. In animals, PLC selectively catalyzes the hydrolysis of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), on the glycerol side of the phosphodiester bond. There is the formation of a weakly enzyme-bound intermediate, inositol 1,2-cyclic phosphodiester, and release of diacyl glycerol (DAG). The intermediate is then hydrolyzed to inositol 1,4,5-trisphosphate (IP3)• Essen, LO; Perisic, O; Katan, M; Yiqin, W; Roberts, MF; Williams, RL (1997).

"Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C". Biochemistry 36 (7): 1704–18.

Phospholipase C.• Thus the two end products are DAG and IP3. The acid/base catalysis

requires two conserved histidine residues and a Ca2+ ion is needed for PIP2 hydrolysis. It has been observed that the active-site Ca2+ coordinates with four acidic residues and if any of the residues are mutated then a greater Ca2+ concentration is needed for catalysis.• Essen, LO; Perisic, O; Katan, M; Yiqin, W; Roberts, MF; Williams, RL

(1997). "Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C". Biochemistry 36 (7): 1704–18.

Phospholipase C.• Receptors that activate this pathway are mainly G protein-coupled receptors

coupled to the Gαq subunit, including:• 5-HT2 serotonergic receptors• α1 (Alpha-1) adrenergic receptors. • Calcitonin receptors• H1 histamine receptors• Metabotropic glutamate receptors, Group I• M1, M3, and M5 muscarinic receptors• Thyroid-Releasing Hormone receptor in anterior pituitary gland

Phospholipase C.• Rhee SG, Choi KD (1992). "Multiple forms of phospholipase C

isozymes and their activation mechanisms". Adv. Second Messenger Phosphoprotein Res. 26: 35–61.PMID 1419362.• DeFranco, Anthony (2008). "Chapter 8: B Lymphocyte Signaling

Mechanisms and Activation". In Paul, William. Fundamental Immunology (Book) (6th ed.). Philadelphia: Lippincott Williams & Wilkins. pp. 270–288. ISBN 0-7817-6519-6.• Downes CP, Michell RH (1981). "The polyphosphoinositide

phosphodiesterase of erythrocyte membranes". Biochem. J. 198 (1): 133–40.PMC 1163219. PMID 6275838

Phospholipase C.• Thompson W and Dawson RMC (1964). "The triphosphoinositide

phosphodiesterase of brain tissue". Biochem. J. 91 (2): 237–243. PMC 1202878.PMID 4284484

Phospholipase C.• Neutrophils are critical inflammatory cells that cause tissue damage in a range of diseases

and disorders. Being bone marrow-derived white blood cells, they migrate from the bloodstream to sites of tissue inflammation in response to chemotactic signals and induce inflammation by undergoing receptor-mediated respiratory burst and degranulation.

• However, the mechanisms that control neutrophil degranulation are not well understood. Recent observations indicate that granule release from neutrophils depends on activation of intracellular signalling pathways, including β-arrestins, the Rho guanosine triphosphatase Rac2, soluble NSF attachment protein (SNAP) receptors, the src family of tyrosine kinases, and the tyrosine phosphatase MEG2.

• Mechanisms of Degranulation in Neutrophils• Paige Lacy • Allergy Asthma Clin Immunol. 2006; 2(3): 98–108.• Published online 2006 Sep 15. doi: 10.1186/1710-1492-2-3-98

Phospholipase C.• Neutrophils contain at least four different types of granules: (1)

primary granules, also known as azurophilic granules; (2) secondary granules, also known as specific granules; (3) tertiary granules; and (4) secretory vesicles.• Mechanisms of Degranulation in Neutrophils• Paige Lacy • Allergy Asthma Clin Immunol. 2006; 2(3): 98–108.• Published online 2006 Sep 15. doi: 10.1186/1710-1492-2-3-98

Phospholipase C.• The primary granules are the main storage site of the most toxic

mediators, including elastase, myeloperoxidase, cathepsins, and defensins. The secondary and tertiary granules contain lactoferrin and matrix metalloprotease 9 (also known as gelatinase B), respectively, among other substances.• Granules are prevented from being released until receptors in the

plasma membrane or phagosomal membrane signal to the cytoplasm to activate their movement to the cell membrane for secretion of their contents by degranulation. This is an important control mechanism as the neutrophil is highly enriched in tissue-destructive proteases.

Phospholipase C.• Mechanisms of Degranulation in Neutrophils• Paige Lacy• Allergy Asthma Clin Immunol. 2006; 2(3): 98–108.• Published online 2006 Sep 15. doi: 10.1186/1710-1492-2-3-98• Translocation and exocytosis of granules in neutrophils require, as a minimum,

increases in intracellular Ca2+, as well as hydrolysis of adenosine triphosphate (ATP) and guanosine triphosphate (GTP). The target molecules for these effectors are numerous and include Ca2+-binding proteins such as annexins and calmodulin and GTP-binding proteins such as G proteins and small monomeric proteins. ATP is used by ATP-hydrolyzing enzymes (adenosine triphosphatases) and kinases, which act by phosphorylating downstream effector molecules.

Phospholipase C.• Increases in intracellular Ca2+ alone are sufficient to induce the release

of many of the granule types in neutrophils, particularly if the concentration of Ca2+ is elevated to sufficiently high levels by the use of Ca2+ionophores such as A23187 or ionomycin. A hierarchy of granule release exists in response to elevating concentrations of Ca2+.

• Mechanisms of Degranulation in Neutrophils• Paige Lacy• Allergy Asthma Clin Immunol. 2006; 2(3): 98–108.• Published online 2006 Sep 15. doi: 10.1186/1710-1492-2-3-98

Phospholipase C.• The release of each type of granule appears to be regulated by different

intracellular signalling pathways. Many neutrophil receptors activate increased Ca2+ levels, including the seven transmembrane-spanning G protein-coupled receptors, such as the formyl peptide receptor (that binds to the bacterial tripeptide f-Met-Leu-Phe) and chemokine receptors (such as CXCR1). Although Ca2+ is a crucial second messenger in the activation of exocytosis, the specific target molecules for Ca2+ in neutrophil degranulation have not yet been identified.• Mechanisms of Degranulation in Neutrophils• Paige Lacy• Allergy Asthma Clin Immunol. 2006; 2(3): 98–108.• Published online 2006 Sep 15. doi: 10.1186/1710-1492-2-3-98

Phospholipase C.• Numerous studies have indicated a role for phospholipids, particularly

polyphosphoinositides, in the regulation of neutrophil degranulation. Polyphosphoinositide production, such as phosphatidylinositol bisphosphate (PIP2), induced by activation of the hematopoietic cell-specific isoform phosphatidylinositol 3-kinase (PI3K)-γ, has been shown to be required for granule exocytosis in permeabilized neutrophil-like cells, HL-60 cells.• ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-

depleted HL60 cells by promoting PIP2 synthesis. Fensome A, Cunningham E, Prosser S, Tan SK, Swigart P, Thomas G, Hsuan J, Cockcroft S• Curr Biol. 1996 Jun 1; 6(6):730-8.•

Phospholipase C.• The intracellular sites of PIP2 formation in neutrophils are not known, but it is likely

to occur both at the plasma membrane and on granule membranes. Regions of PIP2 enrichment in the membrane form essential binding sites for many intracellular signalling molecules, particularly those that contain pleckstrin homology domains. Phosphatidylinositol transfer protein has been shown to be essential for the transport of phosphatidylinositol to cellular membranes as a substrate for PI3K activity to generate PIP2 and is also capable of restoring exocytotic responses in HL-60 cells.• ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-

depleted HL60 cells by promoting PIP2 synthesis.Fensome A, Cunningham E, Prosser S, Tan SK, Swigart P, Thomas G, Hsuan J, Cockcroft S• Curr Biol. 1996 Jun 1; 6(6):730-8.

Phospholipase C.• In addition, a role for phospholipase D has been indicated in

neutrophil degranulation, particularly for primary and secondary granule release, as its product, phosphatidic acid, induces the release of these granules . • Contribution of phopholipase D and a brefeldin A-sensitive ARF to

chemoattractant-induced superoxide production and secretion of human neutrophils. Káldi K, Szeberényi J, Rada BK, Kovács P, Geiszt M, Mócsai A, Ligeti E• J Leukoc Biol. 2002 Apr; 71(4):695-700.

Phospholipase C.• These recent experimental observations reveal that a large group of

intracellular signalling molecules exists to regulate translocation of granules to the cell membrane for docking and fusion to release their contents. Many of these molecules are already natural targets for bacterial toxins to inhibit their function, which highlights their important role in regulating bactericidal mediator release.• Mechanisms of Degranulation in Neutrophils• Paige Lacy• Allergy Asthma Clin Immunol. 2006; 2(3): 98–108.• Published online 2006 Sep 15. doi: 10.1186/1710-1492-2-3-98

Phospholipase C.• T cell antigen receptor (TCR) and natural killer group 2, member D (NKG2D) are two crucial

receptors for T cell cytotoxicity. Compelling evidences suggest that T cell cytotoxicity is TCR-dependent and can be co-stimulated by NKG2D. However, the molecular mechanism of underlying TCR- dependent activation of T cells remains unclear. In this study we demonstrated that TCR but not NKG2D engagement induced lytic granule polarization and promoted T cell cytotoxicity. TCR activation alone was sufficient to trigger Vav1-dependent phospholipase C-1 signaling, resulting in lytic granule polarization and effective killing, whereas NKG2D engagement alone failed to trigger cytotoxicity-related signaling to overcome the inhibitory effect of Cbl-b; therefore, NKG2D engagement alone could not induce effective killing.

• Vav1-phospholipase C-1 (Vav1-PLC-1) Pathway Initiated by T Cell Antigen Receptor (TCR) Activation Is Required to Overcome Inhibition by Ubiquitin Ligase Cbl-b during T Cell Cytotoxicity.

• Shanshan Yin, Jianmin Zhang, Yujia Mao, Yu Hu, Lianxian Cui, Ning Kang1 , and Wei He2 From the Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Peking Union Medical College, National Key Laboratory of Medical Molecular Biology, Beijing 100005, China

Phospholipase C.• . The molecular mechanisms underlying these cytotoxic effects are not known in

detail, although previous studies have pointed to a possible involvement of phosphocholine (PCho), the phosphatidylcholine (PC) head group, as a Ca+2-dependent receptor for PFN and to a role of free PCho, product of phospholipase-mediated PC hydrolysis, as a possible inhibitor of isolated granules' cytotoxicity . Hydrolysis of PC, the major phospholipid in eukaryotic cell membranes, involves distinct classes of phospholipases, including phospholipases A2 (PLA2), C (PC-PLC) and D (PLD).• Expression and role of phosphatidylcholine-specific phospholipase C in human

NK and T lymphocyte subsets. Francesca Spadaro1 , Serena Cecchetti1 , Massimo Sanchez1 , Clara Maria Ausiello2 , Franca Podo1 and Carlo Ramoni1• Eur. J. Immunol. 2006. 36: 3277–3287

Phospholipase C.• , a high expression of PC-PLC enzyme on the outer membrane surface

seems to be an important feature of NK cells, in which increased enzyme externalization is apparently correlated with cell maturation and linked to cytolytic activity. The association of this enzyme with PFN molecules in the immunological synapse points to a common final step of the cytolytic machinery in cytotoxic CD8+ T lymphocytes and in NK cells.• Expression and role of phosphatidylcholine-specific phospholipase C in

human NK and T lymphocyte subsets. Francesca Spadaro1 , Serena Cecchetti1 , Massimo Sanchez1 , Clara Maria Ausiello2 , Franca Podo1 and Carlo Ramoni1• Eur. J. Immunol. 2006. 36: 3277–3287

Phospholipase C.• http://www.slideshare.net/dlpopov/anti-radiation-vaccine-technology• http://www.slideshare.net/dlpopov/radiation-toxinspossible-new-clas

s-of-anti-cancer-drugs-comparison-of-anticancer-drugs-by-mechanisms-of-action• http://www.slideshare.net/dlpopov/radiation-protection-anti-radiatio

n-vaccine-technology• http://www.slideshare.net/dlpopov/radiation-protection-antigen-pres

enting-cells

Phospholipase C.• http://www.slideshare.net/dlpopov/radiation-cytotoxicity• http://www.slideshare.net/dlpopov/leukocyte-and-lymphocyte-cytot

oxicity• Blood. 2013 Jun 6;121(23):4672-83. doi: 10.1182/blood-2012-08-

453738. Epub 2013 Apr 30.• LAMP1/CD107a is required for efficient perforin delivery to lytic

granules and NK-cell cytotoxicity.• Krzewski K1, Gil-Krzewska A, Nguyen V, Peruzzi G, Coligan JE.

Phospholipase C. Lysosome-associated membrane protein (LAMP) .• Secretory lysosomes of natural killer (NK) cells, containing perforin and granzymes,

are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells.• Blood. 2013 Jun 6;121(23):4672-83. doi: 10.1182/blood-2012-08-453738. Epub

2013 Apr 30.• LAMP1/CD107a is required for efficient perforin delivery to lytic granules and

NK-cell cytotoxicity.• Krzewski K1, Gil-Krzewska A, Nguyen V, Peruzzi G, Coligan JE.

Phospholipase C. Lysosome-associated membrane protein (LAMP) .• Reduction of LAMP1 expression affects the movement of lytic granules and results in

decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network-derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity.

• Blood. 2013 Jun 6;121(23):4672-83. doi: 10.1182/blood-2012-08-453738. Epub 2013 Apr 30.• LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell

cytotoxicity.• Krzewski K1, Gil-Krzewska A, Nguyen V, Peruzzi G, Coligan JE.

Phospholipase C. Lysosome-associated membrane protein (LAMP) .• http://www.slideshare.net/dlpopov/anti-radiation-antidote-inhibition

-and-neutralization-of-radiation-toxicity-with-therapeutic-monoclonal-antibodies

Phospholipase C.• Santa Cruz Biotechnology, Inc. offers a broad range of LAMP antibodies. Select

LAMP antibodies from several monoclonal and/or polyclonal LAMP antibodies listed below. View detailed LAMP antibody specifications by linking to the specific product blocks.• http://www.scbt.com/table-lamp.html• http://www.biolegend.com/apc-anti-human-perforin-antibody-4001.html• Perforin is a 70 kD cytolytic protein that is expressed in the cytoplasmic

granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Perforin is one of the major effector molecules used by cytotoxic T cells and NK cells to mediate targeted cell lysis.• Biolegend Inc.

Phospholipase C.• Recent experimental observations reveal that a large group of

intracellular signalling molecules acting to regulate translocation of granules to the cell membrane for docking and fusion to release their contents. Many of these molecules are a targets for therapeutic polyclonal or monoclonal antibodies, which can inhibit their function, and highlights their important role in regulating tissue damaging by mediator release after irradiation.• Anti Radiation Antitoxin.• Anti Radiation Vaccine.