Radiation Protection: Phospholipase C, LAMP and Phospholipase C, LAMP inhibition.

1. Radiation Protection:
Phospholipase C,LAMP and
Phospholipase C, LAMP
inhibition.
Dmitri Popov, PhD, Radiobiology. Canada.
MD (Russia).

2. 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

3. 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

4. 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

5. 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.

6. 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.

7. 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

8. 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

9. Phospholipase C.
• Thompson W and Dawson RMC (1964). "The triphosphoinositide
phosphodiesterase of brain tissue". Biochem. J. 91 (2): 237–
243. PMC 1202878.PMID 4284484

10. 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

11. 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

12. 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.

13. 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.

14. 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

15. 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

16. 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.
•

17. 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.

18. 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.

19. 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

20. 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

21. 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

22. 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

23. Phospholipase C.
• http://www.slideshare.net/dlpopov/anti-radiation-vaccine-
technology
• http://www.slideshare.net/dlpopov/radiation-toxinspossible-new-
class-of-anti-cancer-drugs-comparison-of-anticancer-drugs-by-
mechanisms-of-action
• http://www.slideshare.net/dlpopov/radiation-protection-anti-
radiation-vaccine-technology
• http://www.slideshare.net/dlpopov/radiation-protection-antigen-
presenting-cells

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

25. 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.

26. 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.

27. 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

28. 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.

29. 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.