Phospholipase A

1. Radiation Protection:
Phospholipase A.
Dmitri Popov. PhD Radiobiology, MD (Russia),
Advanced MedicalTechnology and Systems Inc. Canada.

2. Phospholipase A.
• Research Proposal: Radiation Protection : Phospholipase A.
• Dmitri Popov
• Full-text Research Proposal · Mar 2016
• Add resources
• File name: RadiationProtectionPhospholipaseA.pptx
DOI: 10.13140/RG.2.1.4878.7445

3. Phospholipase A
• Key words: phospholipase A2, phospholipase A2 receptor (PLA2R),
• Phospholipase C.

4. Phospholipase A.
• The present research relates generally to the fields of radiation biology,
toxicology and medicine.
• The blocking antibodies reduce effects of ionizing radiation and protect
cells and reduce toxic effects of radiation.

5. Phospholipase A.
• A phospholipase is an enzyme that hydrolyzes phospholipids into fatty acids and
other lipophilic substances.There are four major classes, termed A, B, C and D,
distinguished by the type of reaction which they catalyze:
• Phospholipase A
• Phospholipase A1 - cleaves the SN-1 acyl chain.
• Phospholipase A2 - cleaves the SN-2 acyl chain, releasing arachidonic acid.
• Phospholipase B - cleaves both SN-1 and SN-2 acyl chains; this enzyme is also
known as a lysophospholipase.

6. Phospholipase A.
• PhospholipaseC - cleaves before the phosphate, releasing diacylglycerol and a phosphate-
containing head group. PhospholipaseCs play a central role in signal transduction,
releasing the second messenger inositol triphosphate.
• Phospholipase D - cleaves after the phosphate, releasing phosphatidic acid and an alcohol.
• Types C and D are considered phosphodiesterases.
• PhospholipaseA2 acts on the intact lecithin molecule and hydrolyses the fatty acid
esterified to the second carbon atom.The resulting products are lysolecithin and a fatty
acid. PhospholipaseA2 is an enzyme present in the venom of bees and viper snakes

7. Heckmann LH, Sibly RM,Timmermans MJ, Callaghan A - Front. Zool. (2008)

8. Citation: AntiviralTherapy 2012; 17:485-493
doi: 10.3851/IMP1990

9. Dingzhi Wang & Raymond N. DuBois
Nature Reviews Cancer 10, 181-193 (March 2010)
doi:10.1038/nrc2809

10. [Mediator function of molecular factors--lipoxygenase enzyme systems during exposure
to ionizing radiation].
Budnitskaia EV. Radiobiologiia. 1986 Jul-Aug;26(4):435-46.
• “review of the national literature on the mediatory function of lipoxygenase systems affected by ionizing
radiation, reports data on the functional role of lipoxygenases, their regulator--mediate contribution to
radiation response of plants and animals.The latest data are submitted concerning the biological
regulatory systems, eicosanoids--leukotrienes, formed under the effect of lipoxygenases which catalyse
oxidation of polyunsaturated arachidonic acid (20:4).The structure and function of leukotrienes are
described for these are necessary in studying the biochemical functions of lipoxygenases after ionizing
irradiation. Emphasis is made on biologically active leukotrienes which take part in biological processes
involved in inflammatory and hypersensitive reactions under the effect of radiation. Possible involvement
of the lipoxygenase systems in metabolism regulation, its resistance and activation in a living body during
development of radiopathological processes.The modelling concepts are considered for perspective
radiation research aimed at biotechnological utilisation of lipoxygenases. Some regularities of mediatory
function of lipoxygenase systems have been found.An assumption has been made that lipoxygenases and
leukotrienes play an important role in the life of irradiated cells and tissues.”

11. SnakeVenom Phospholipases A, Jeanne F’rigent-
Dachary From the Laboratoire d’Hematologie,
Universite de Bordeaux II, France
• “Some purified phospholipases A2 originating from different snake venoms
have been shown to possess anticoagulant properties . It appears that they
inhibit blood coagulation via their action on phospholipids . It is well known
that certain phospholipids accelerate clot formation by providing a surface
on which the clotting factors interact “

12. SnakeVenom Phospholipases A, Jeanne F’rigent-
Dachary From the Laboratoire d’Hematologie,
Universite de Bordeaux II, France
• Anticoagulant phospholipases are able to bind both classes of phospholipids
whatever their fluidities.Therefore in usual mixtures of pro-coagulant lipids,
i.e. PC-PS binary systems, these phospholipases can interact with all lipids
and compete with clotting factors for the same interface area. Furthermore,
these phospholipases hydrolyze both classes of lipids, then the degraded
interface can no longer serve as pro-coagulant surface.

13. https://en.wikipedia.org/wiki/Blocking_antibod
y
• A blocking antibody is an antibody that does not have a reaction when
combined with an antigen, but prevents other antibodies from combining
with that antigen.
• This function of blocking antibodies has had a variety of clinical and
experimental uses.
• The term can also be used for inhibiting antibody, prozone
phenomenon and, agglutination reaction.

14. Phospholipase A2 or Phospholipase A2
membrane receptorsAntibodies.
Inhibition of protein kinases blocks radiation-mediated gene induction and
prevent radiation induced apoptosis (Uckun et al., 1992), necrosis and
increase radiation survival (Hallahan et al., 1992)).

15. Photochem Photobiol. 1993 Feb;57(2):383-90.
Ultraviolet radiation-induced phospholipase A2 activation occurs in
mammalian cell membrane preparations.
Cohen D1, DeLeoVA.
• Ultraviolet erythema in human skin is mediated in part by membrane derivatives of
arachidonic acid (AA). UVA (320-400 nm) and UVB (290-320 nm) have been shown
to induce release of AA from intact mammalian cells in culture. In order to
investigate the mechanism of this release we examined the effect of UVA and UVB
on release of [3H] AA from membrane preparations of murine fibroblasts. C3H
10T1/2 cells were prelabelled for 24 h with [3H] AA.The membrane fractions of the
cells were separated after lysis by differential centrifugation.The membranes were
irradiated in suspension and the [3H] AA released from the membranes was
determined by scintillation spectroscopy of supernatants 3-4 h after irradiation.

16. Photochem Photobiol. 1993 Feb;57(2):383-90.
Ultraviolet radiation-induced phospholipase A2 activation occurs in
mammalian cell membrane preparations.
Cohen D1, DeLeoVA.
• Both UVA and UVB induced release of AA from the membrane preparations.The
response to UVB was small but significant, reaching levels approximately 150% of
control release at doses of 1,200-4,000 J/m2.The response to UVA was larger;
doses of 2.5-5.0 J/cm2 induced release equal to twice control (200%) levels, while
doses of 10-20 J/cm2 induced maximal release at levels approximately 400% of
control.Time course studies with UVB and UVA showed maximal release at 4 h
after irradiation. When the membrane preparations were incubated with a
polyclonal anti-phospholipase A2 antibody the UV induced release of [3H] AA was
completely inhibited in both UVB (1200 J/m2) and UVA (10 J/cm2) treated cells.+

17. Cytosolic phospholipase A2 regulates viability of irradiated vascular endothelium.
Cell Death and Differentiation (2008) 15, 1641–1653; doi:10.1038/cdd.2008.93;
published online 20 June 200. E MYazlovitskaya1,2, A G Linkous1, D KThotala1, K
C Cuneo1 and D E Hallahan1,2,3
• “Radiosensitivity of various normal tissues is largely dependent on radiation-
triggered signal transduction pathways. Radiation simultaneously initiates
distinct signaling from both DNA damage and cell membrane. Specifically,
DNA strand breaks initiate cell-cycle delay, strand-break repair or programmed
cell death, whereas membrane-derived signaling through phosphatidylinositol
3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-
regulated kinase (ERK) enhances cell viability. Activation of cytosolic
phospholipase A2 (cPLA2) and production of the lipid second-messenger
lysophosphatidylcholine were identified as initial events (within 2 min) required
for radiation-induced activation of Akt and ERK1/2 in vascular endothelial
cells.”

18. Cytosolic phospholipase A2 regulates viability of irradiated vascular
endothelium.
Cell Death and Differentiation (2008) 15, 1641–1653; doi:10.1038/cdd.2008.93;
published online 20 June 200. E MYazlovitskaya1,2, A G Linkous1, D KThotala1,
K C Cuneo1 and D E Hallahan1,2,3
• “Inhibition of cPLA2 significantly enhanced radiation-induced
cytotoxicity due to an increased number of multinucleated giant cells
and cell cycle-independent accumulation of cyclin B1 within 24–48 h of
irradiation. Delayed programmed cell death was detected at 72–96 h
after treatment. Endothelial functions were also affected by inhibition of
cPLA2 during irradiation resulting in attenuated cell migration and
tubule formation.”

19. Cytosolic phospholipase A2 regulates viability of irradiated vascular
endothelium.
Cell Death and Differentiation (2008) 15, 1641–1653; doi:10.1038/cdd.2008.93;
published online 20 June 200. E MYazlovitskaya1,2, A G Linkous1, D KThotala1,
K C Cuneo1 and D E Hallahan1,2,3
• “The role of cPLA2 in the regulation of radiation-induced activation of
Akt and ERK1/2 and cell viability was confirmed using human umbilical
vein endothelial cells transfected with shRNA for cPLA2α and cultured
embryonic fibroblasts from cPLA2α−/− mice. In summary, an immediate
radiation-induced cPLA2-dependent signaling was identified that
regulates cell viability and, therefore, represents one of the key
regulators of radioresistance of vascular endothelial cells.”

20. Ionizing Radiation Acts on Cellular Membranes to Generate Ceramide and
Initiate Apoptosis By Adriana Haimovitz-Friedman, Chu-Cheng Kan,* Desiree
Ehleiter, Roger S. Persaud, Maureen McLoughlin, Zvi Fuks, and Richard N.
Kolesnick*
• Recent investigations provided evidence that the sphingomyelin signal
transduction pathway mediates apoptosis for tumor necrosis factor c~
(TNF-o 0 in several hematopoietic and nonhematopoietic ceils. In this
pathway,TNF-receptor interaction initiates sphingomyelin hydrolysis to
ceramide by a sphingomyelinase.

21. Ionizing Radiation Acts on Cellular Membranes to Generate Ceramide and
Initiate Apoptosis By Adriana Haimovitz-Friedman, Chu-Cheng Kan,* Desiree
Ehleiter, Roger S. Persaud, Maureen McLoughlin, Zvi Fuks, and Richard N.
Kolesnick*
• In this pathway,TNF-receptor interaction initiates sphingomyelin hydrolysis
to ceramide by a sphingomyelinase. Ceramide acts as a second messenger
stimulating a ceramide-activated serine/threonine protein kinase.The
present studies show that ionizing radiation, likeTNF, induces rapid
sphingomyelin hydrolysis to ceramide and apoptosis in bovine aortic
endothelial cells. Elevation of ceramide with exogenous ceramide analogues
was su~cient for induction of apoptosis.

22. Ionizing Radiation Acts on Cellular Membranes to Generate Ceramide and
Initiate Apoptosis By Adriana Haimovitz-Friedman, Chu-Cheng Kan,* Desiree
Ehleiter, Roger S. Persaud, Maureen McLoughlin, Zvi Fuks, and Richard N.
Kolesnick*
• Protein kinase C activation blocked both radiation-induced sphingomyelin
hydrolysis and apoptosis, and apoptosis was restored by ceramide
analogues added exogenously. Ionizing radiation acted directly on
membrane preparations devoid of nuclei, stimulating sphingomyelin
hydrolysis enzymatically through a neutral sphingomyelinase.These studies
provide the first conclusive evidence that apoptotic signaling can be
generated by interaction of ionizing radiation with cellular membranes and
suggest an alternative to the hypothesis that direct DNA damage mediates
radiation-induced cell kill.

23. Conclusion
• In conclusion, our opinion suggests that anti-PLA2 antibodies could work as
a specific marker for irradiated mammals. It also indicates that there is a
common pathophysiology among irradiated mammals with Acute Radiation
Syndromes and Chronic Radiation Disease.
• Development of anti-PLA2 antibodies which will work as a sensitive
biomarker may provide new tools such as ELISA for diagnosis, Acute
Radiation Syndromes severity monitoring, and invention of new immune -
therapy strategies in Acute Radiation Syndromes.

24. Literature.
• Biochimica et Biophysica Acta (BBA) - Biomembranes
• Volume 604, Issue 2, 30 September 1980, Pages 191-246
• H.Van Den Bosch
• Intracellular phospholipases A.

25. Literature
• Boffa, M.-C., Rothen, C.,Verheij, H. M.,Verger, R., and de Xaas,
• 2. Boffa, M.-C., and Boffa, G. A. (1976) Biochim. Biophys. Acta
• 3. Papahadjopoulos, D., and Hanahan, D. J. (1964) Biochim.

26. Literature.
• SnakeVenom Phospholipases A, A FLUORESCENCE STUDY OFTHEIR
BINDINGTO PHOSPHOLIPIDVESICLES CORRELATION WITHTHEIR
ANTICOAGULANT ACTIVITIES‘
• Jeanne F’rigent-Dachary From the Laboratoire d’Hematologie, Universite
de Bordeaux II, 33076, Bordeaux Cedex France et al.

27. Literature.
• Carter et al., "Tyrosine phosphorylation of phospholipase C induced by
membrane immunoglobulin in B lymphocytes," Proc. Natl. Acad. Sci. USA,
88:2745-2749, 1991.

28. Literature.
• Hallahan et al., "Molecular Basis for the Use of Glucocorticoids,
Nonsteroidals and Pentoxifylline to Minimize the Acute Effects of
Radiotherapy," Cancer Research, 51:4565-4569, 1991.

29. Literature.
• Hallahan et al., "Tumor Necrosis Factor Gene Expression Is Mediated by
Protein Kinase C following Activation by Ionizing Radiation," Cancer
Research, 51:4565-4569, 1991.

30. Literature.
• Nishizuka, "Intracellular Signaling by Hydrolysis of Phospholipids and
Activation of Protein Kinase C," Science, 258:607-614, 1992.

31. Literature.
• Uckun et al., "Ionizing radiation stimulates unidentified tyrosine-specific
protein kinases in human B-lymphocyte precursors, triggering apoptosis
and clonogenic cell death," Proc. Natl. Acad. Sci. USA, 89:9005-9009, 1992.

32. Literature.
• Uckun et al., "Tyrosine phosphorylation is a mandatory proximal step in
radiation-induced activation of the protein kinase C signaling pathway in
human B-lymphocyte precursors," Proc. Natl. Acad. Sci. USA, 90:252-256,
1993.

33. Literature.
• Prostaglandins and Inflammation
• Emanuela Ricciotti, PhD and GarretA. FitzGerald, MD.* Institute for
Translational Medicine andTherapeutics, University of Pennsylvania,
Philadelphia, Pa.
• Published in final edited form as: ArteriosclerThrombVasc Biol. 2011 May ;
31(5): 986–1000. doi:10.1161/ATVBAHA.110.207449.

34. Literature.
• Cytosolic phospholipase A2 regulates viability of irradiated vascular
endothelium.
Cell Death and Differentiation (2008) 15, 1641–1653;
doi:10.1038/cdd.2008.93; published online 20 June 200. E MYazlovitskaya1,2,
A G Linkous1, D KThotala1, K C Cuneo1 and D E Hallahan1,2,3

35. Literature.
• Kolesnick R, Fuks Z. Radiation and ceramide-induced apoptosis. Oncogene
2003; 22: 5897–5906.

36. Literature.
• . Haimovitz-Friedman A, Kan CC, Ehleiter D, Persaud RS, McLoughlin M,
Fuks Z et al. Ionizing radiation acts on cellular membranes to generate
ceramide and initiate apoptosis. J Exp Med 1994; 180: 525–535.

37. Literature.
• Valerie K,YacoubA, Hagan MP, Curiel DT, Fisher PB, Grant S et al. Radiation-
induced cell signaling: inside-out and outside-in. Mol CancerTher 2007; 6:
789–801.

38. Literature.
• FarooquiAA, Horrocks LA. Phospholipase A2-generated lipid mediators in
the brain: the good, the bad, and the ugly. Neuroscientist 2006; 12: 245–
260.

39. Literature.
• HirabayashiT, MurayamaT, ShimizuT. Regulatory mechanism and
physiological role of cytosolic phospholipase A2. Biol Pharm Bull 2004; 27:
1168–1173.