Lipoxygenase regulation in vivo and in vitro by lipid compounds
Lipoxygenases (LOs) are known as one of the enzymes of lipid peroxidation. The majority of LOs are soluble enzymes and have affinity to membranes. The enzyme translocation from a cytosol to a membrane surface is one of the stages of regulation of the amount of LO catalysis products in the cell. A so...
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Skaterna, T.D. Kopich, V.M. Kharitonenko, G.I. Kharchenko, O.V. 2019-06-11T17:29:06Z 2019-06-11T17:29:06Z 2015 Lipoxygenase regulation in vivo and in vitro by lipid compounds / T.D. Skaterna, V.M. Kopich, G.I. Kharitonenko, O.V. Kharchenko // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 161-173. — Бібліогр.: 77 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008DC https://nasplib.isofts.kiev.ua/handle/123456789/152449 577.152.1 Lipoxygenases (LOs) are known as one of the enzymes of lipid peroxidation. The majority of LOs are soluble enzymes and have affinity to membranes. The enzyme translocation from a cytosol to a membrane surface is one of the stages of regulation of the amount of LO catalysis products in the cell. A sorption to the membrane surface is described for most LOs from plant and animal sources. This review presents the data about regulation of the LO activity by the lipid compounds – both natural and chemically modified. Lipids might regulate the LO activity through: protein-lipid interactions of C2 domain with the membrane, changes in the enzyme affinity, the LOs translocation, allosteric regulation, increase in the selectivity towards substrates. The regulatory effect of active compound on the enzyme activity depends on the lipophilicity of effectors. Considering the LO activity it is necessary to take into account the enzyme microenvironment and its influence on the range of the LO products. Ліпоксигенази (ЛО) відомі як одні з ферментів перекисного окислення ліпідів. Більшість ЛО є розчинними ферментами та характеризуються аффіністю до мембран. Транслокація ферменту з цитозолю на мембранну поверхню одна з стадій регуляції рівня продуктів ліпоксигеназного каталізу у клітині. Сорбція на мембранну поверхню описана для більшості ЛОз з рослинних та тваринних джерел. Даний огляд представляє дані по регуляції ЛОз активності природніми та хімічно модифікованими речовинами ліпідної природи. Здатність ліпідів регулювати ЛО активність може здійснюватись через: білок-ліпідні взаємодії C2 домену з мембраною, зміну аффінності ферменту, транслокацію ЛОз, алостеричну регуляцію, збільшення селективності до субстрату. Регуляторний вплив активної сполуки на ферментативну активність залежить від рівня ліпофільності ефекторів. При поясненні ліпоксигеназного каталізу необхідно враховувати вплив мікрооточення ферменту на рівень ЛО. Липоксигеназы (ЛО) известны как одни из ферментов перекисного окисления липидов. Большинство ЛО растворимые ферменты и характеризуются аффиностью к мембранам. Транслокация фермента из цитозоля на мембранную поверхность одна из стадий регуляции уровня продуктов липоксигеназного катализа в клетке. Сорбция на мембранную поверхность описана для большинства ЛОз из растительных и животных источников. Данный обзор представляет данные по регуляции ЛО активности природными и химически модифицированными соединениями липидной природы. Возможность липидов регулировать ЛО активность может осуществляться через: белок-липидные взаимодействия C2 домена с мембраной, изменение аффинности фермента, транслокацию ЛОз, аллостерическую регуляцию, селективность к типу субстрата. Регуляторное влияние активного соединения на ферментативную активность зависит от уровня липофильности еффекторов. При объяснении липоксигеназного катализа необходимо учитывать влияние микроокружения фермента на уровень ЛО продуктов. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Reviews Lipoxygenase regulation in vivo and in vitro by lipid compounds Регуляція ліпоксигеназ in vivo та in vitro сполуками ліпідної природи Регуляция липоксигеназ in vivo и in vitro соединениями липидной природы Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Lipoxygenase regulation in vivo and in vitro by lipid compounds |
| spellingShingle |
Lipoxygenase regulation in vivo and in vitro by lipid compounds Skaterna, T.D. Kopich, V.M. Kharitonenko, G.I. Kharchenko, O.V. Reviews |
| title_short |
Lipoxygenase regulation in vivo and in vitro by lipid compounds |
| title_full |
Lipoxygenase regulation in vivo and in vitro by lipid compounds |
| title_fullStr |
Lipoxygenase regulation in vivo and in vitro by lipid compounds |
| title_full_unstemmed |
Lipoxygenase regulation in vivo and in vitro by lipid compounds |
| title_sort |
lipoxygenase regulation in vivo and in vitro by lipid compounds |
| author |
Skaterna, T.D. Kopich, V.M. Kharitonenko, G.I. Kharchenko, O.V. |
| author_facet |
Skaterna, T.D. Kopich, V.M. Kharitonenko, G.I. Kharchenko, O.V. |
| topic |
Reviews |
| topic_facet |
Reviews |
| publishDate |
2015 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Регуляція ліпоксигеназ in vivo та in vitro сполуками ліпідної природи Регуляция липоксигеназ in vivo и in vitro соединениями липидной природы |
| description |
Lipoxygenases (LOs) are known as one of the enzymes of lipid peroxidation. The majority of LOs are soluble enzymes and have affinity to membranes. The enzyme translocation from a cytosol to a membrane surface is one of the stages of regulation of the amount of LO catalysis products in the cell. A sorption to the membrane surface is described for most LOs from plant and animal sources. This review presents the data about regulation of the LO activity by the lipid compounds – both natural and chemically modified. Lipids might regulate the LO activity through: protein-lipid interactions of C2 domain with the membrane, changes in the enzyme affinity, the LOs translocation, allosteric regulation, increase in the selectivity towards substrates. The regulatory effect of active compound on the enzyme activity depends on the lipophilicity of effectors. Considering the LO activity it is necessary to take into account the enzyme microenvironment and its influence on the range of the LO products.
Ліпоксигенази (ЛО) відомі як одні з ферментів перекисного окислення ліпідів. Більшість ЛО є розчинними ферментами та характеризуються аффіністю до мембран. Транслокація ферменту з цитозолю на мембранну поверхню одна з стадій регуляції рівня продуктів ліпоксигеназного каталізу у клітині. Сорбція на мембранну поверхню описана для більшості ЛОз з рослинних та тваринних джерел. Даний огляд представляє дані по регуляції ЛОз активності природніми та хімічно модифікованими речовинами ліпідної природи. Здатність ліпідів регулювати ЛО активність може здійснюватись через: білок-ліпідні взаємодії C2 домену з мембраною, зміну аффінності ферменту, транслокацію ЛОз, алостеричну регуляцію, збільшення селективності до субстрату. Регуляторний вплив активної сполуки на ферментативну активність залежить від рівня ліпофільності ефекторів. При поясненні ліпоксигеназного каталізу необхідно враховувати вплив мікрооточення ферменту на рівень ЛО.
Липоксигеназы (ЛО) известны как одни из ферментов перекисного окисления липидов. Большинство ЛО растворимые ферменты и характеризуются аффиностью к мембранам. Транслокация фермента из цитозоля на мембранную поверхность одна из стадий регуляции уровня продуктов липоксигеназного катализа в клетке. Сорбция на мембранную поверхность описана для большинства ЛОз из растительных и животных источников. Данный обзор представляет данные по регуляции ЛО активности природными и химически модифицированными соединениями липидной природы. Возможность липидов регулировать ЛО активность может осуществляться через: белок-липидные взаимодействия C2 домена с мембраной, изменение аффинности фермента, транслокацию ЛОз, аллостерическую регуляцию, селективность к типу субстрата. Регуляторное влияние активного соединения на ферментативную активность зависит от уровня липофильности еффекторов. При объяснении липоксигеназного катализа необходимо учитывать влияние микроокружения фермента на уровень ЛО продуктов.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152449 |
| citation_txt |
Lipoxygenase regulation in vivo and in vitro by lipid compounds / T.D. Skaterna, V.M. Kopich, G.I. Kharitonenko, O.V. Kharchenko // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 161-173. — Бібліогр.: 77 назв. — англ. |
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161
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 3. P. 161–173
doi: http://dx.doi.org/10.7124/bc.0008DC
Reviews
UDC 577.152.1
Lipoxygenase regulation in vivo and in vitro by lipid compounds
T. D. Skaterna, V. M. Kopich, G. I. Kharitonenko, O. V. Kharchenko
Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine
1, Murmans'ka Str., Kyiv, Ukraine, 02660
skaterna.t@ukr.net
Lipoxygenases (LOs) are known as one of the enzymes of lipid peroxidation. The majority of LOs are
soluble enzymes and have affi nity to membranes. The enzyme translocation from a cytosol to a mem-
brane surface is one of the stages of regulation of the amount of LO catalysis products in the cell. A sorp-
tion to the membrane surface is described for most LOs from plant and animal sources. This review
presents the data about regulation of the LO activity by the lipid compounds – both natural and chemi-
cally modifi ed. Lipids might regulate the LO activity through: protein-lipid interactions of C2 domain
with the membrane, changes in the enzyme affi nity, the LOs translocation, allosteric regulation, increase
in the selectivity towards substrates. The regulatory effect of active compound on the enzyme activity
depends on the lipophilicity of effectors. Considering the LO activity it is necessary to take into account
the enzyme microenvironment and its infl uence on the range of the LO products.
K e y w o r d s: lipoxygenase, allosteric regulation, phospholipids, inhibition, activation.
© 2015 T. D. Skaterna et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Biological signifi cance of the lipoxygenase pathway
products in living organisms explains an interest
paid to the research on regulation mechanisms of
this key enzyme and a possibility to correct the level
of lipoxygenase metabolites. Lipoxygenases (oxi-
doreductase, EC 1.13.11.- ; LO) are the enzymes of
lipid metabolism, which catalyze the oxygen inser-
tion into the 1,4-cis,cis-pentadiene fragment of poly-
unsaturated fatty acids (PUFAs) with production of
the corresponding hydroperoxide derivatives (HP).
The LO products of PUFAs oxidation pathways are
diverse signal compounds, animals leukotrienes and
lipoxynes, coral prostaglandin-like LO metabolites,
lactones of microorganisms, plant jasmonates, etc.
They are involved in the apoptosis and cell prolifera-
tion, metabolism and transportation, cell-cell inter-
actions and infl ammatory processes. The quantita-
tive and qualitative composition of LO metabolites
changes at various pathological conditions of ani-
mal, human and plant cells during the adaptation to
environmental factors and at the conditions of in-
tense growth and development. This is why the fi nd-
ing of new substances, which can regulate the LO
activity, is a task of urgent importance.
The factors potentially able to regulate the 5-LO
activity are as follows: 1 – factors changing the en-
zyme activity by facilitating the substrate accessibili-
ty; 2 – Fe2+oxidation to Fe3+active state; 3 – stabiliza-
tion of the active 5-LO-form. Ions Ca2+, Mg2+, phos-
pholipids, glycerol, membranes, ATP are capable of
particular increasing the 5-LO activity. The hydroper-
oxides level also determines the 5-LO activity and
regulates the redox state of ferrum in the active site.
Posphorylation and interaction with the protein fac-
tors from polymorphonuclear leukocytes (coactosin-
like protein up-regulates the Ca2+-induced 5-LO activ-
ity [1]) and the membrane associated protein FLAP
(5-LO activating protein bounds with a nuclear mem-
brane [2]) are related to the factors, which are able to
control the enzyme activity in the cell.
162
T. D. Skaterna, V. M. Kopich, G. I. Kharitonenko, O. V. Kharchenko
In cells, 5-LO locates in a soluble form in cytosol
(eosinophils, neutrophils, macrophages) or in a nu-
clear soluble compartment associated with chroma-
tin (alveolar macrophages, Langerhans cells) [3-4].
In plants, the main pool of 13-LO products associ-
ates with inner and external plastids membranes
[5–7]; 13-hydroperoxide lyase and allene oxide syn-
thase are the enzymes, which utilize the products of
13-LO oxidation of linoleic (LA) and linolenic acids
as the substrates and are also connected with inner
and external plastids membranes respectively. The
source of 9-hydroperoxides synthesis is predomi-
nantly the plant cytoplasmic membrane [5]. It was
suggested that there is a connection between the LO
activity of microsomal and mitochondrial mem-
branes, but the data presented are insuffi cient [7–8].
5-LO is also associated with the lipid rafts with pro-
teinkinase C II and other rafts proteins which were
demonstrated in the mantle cell lymphoma [9].
The substrates of LO reaction are polyunsaturated
fatty acids that are a part of membrane phospholip-
ids. The majority of LO are soluble enzymes and
have affi nity to membranes. Under cell stimulation,
5-LO migrates to the nuclear membrane where an-
other enzyme phospholipase A2 liberates arachidonic
acid (AA) from phospholipids during the reaction,
which is utilized by 5-LO as a substrate. The mem-
brane associated protein FLAP facilitates the sub-
strate transport to 5-LO [10], which is connected
with the nuclear membrane by amino acids. This
protein can be phosphorylated by MAPK-activated
proteinkinase (MAPKAPK)-2 and ERKs. Thus,
FLAP controles the 5-LO products synthesis.
1. Regulation of lipoxygenase
activity by cell membrane compounds
The main part of the LO reaction substrates, polyun-
saturated fatty acids, is located in the complexes
com posed of membranes or in the lipoprotein com-
plexes. PUFAs are insoluble within the range of pH
physiological values for most LO. pHopt is more al-
kaline for 13-LO, and this type of LO does not re-
quire an interaction with the membrane surface for
active transition. The enzyme translocation from cy-
tosol to the membrane surface is one of the stages of
regulation of the amount of LO catalysis products in
the cell. A sorption to the membrane surface is de-
scribed for most LO of plant and animal sources.
In animal cells the leukotrienes biosynthesis starts
with the 5-LO translocation from cytosol to the nu-
clear membrane surface. Phospholipase A2 liberates
AA and thus it is the forerunner to LO. The next en-
zyme in transforming HP LTA4 synthase is also con-
nected with the nuclear membrane [11]. It is assumed
that the 5-LO exclusive sorption on the nuclear me-
m brane surface depended on the membrane associa-
ted protein FLAP (fi ve-lipoxygenase activating pro-
tein) [12] and a high affi nity to zwitterionic phospha-
tidylcholine (PC) [24, 26], which is a dominant com-
ponent of the nuclear membrane. FLAP also has a
high affi nity to the enzyme and PUFAs. Due to these
properties, FLAP not only provides the enzyme as-
sociation with specifi c membrane component but also
regulates the enzyme interactions with the substrate
creating local clusters of a high PUFAs concentra-
tion. The sequence of FLAP is 31 % identical to the
microsomal LTC4 synthase [13]. FLAP can be asso-
ciated with the endoplasmatic reticulum and lipid
rafts, as the 5-LOX association with lipid rafts in
man tle cell lymphoma was established [9]. In te res-
tin gly, FLAP was shown to be a part of secretory
vesicles from the human neutrophils and exosomes
of human monocyte-derived macrophages or mono-
cyte-derived dendritic cells with LTA4 hydrolase and
LTC4 synthase [14]. Probably, the complex of 5-LO,
FLAP, LTA4 hydrolase and LTC4 synthase with the
membrane vesicles can transfer out of the cell and
the reaction products of this complex regulate the
metabolism outside as compounds of distant action.
Possibly, the mechanism for LO from other sources
is similar, which is confi rmed by the activation of
potato tuber 5-LO in the presence of FLAP [15].
5-LO demonstrates the activity at the interface
lipid:water. The reaction runs similarly to phospholi-
pase A2. The heterophase nature of the reaction is
determined by the presence in the enzymes structure
of C2-like domain - N-terminal domain consisting of
eight antiparallel β-sheets. This domain has some
homology of its structure and function with C2 do-
main of phospholipases and proteinkinase C [17–19].
163
Lipoxygenase regulation in vivo and in vitro by lipid compounds
Similarity of mechanisms of these interactions pro-
vides a strong evidence of their universality in reg-
ulating the activity of key enzymes of the signaling
systems. The residues in the ligand binding loops
of β-sandwich bind Ca2+, cellular membranes, and
co ac tosin-like protein (CLP) [4, 21–22]. C2 domain
is characteristic of the association of membrane
phospholipids and this process can be mediated by
Ca2+ ions [23].
The interaction with membrane surface was de-
scribed for the cloned 5-LO from leucocytes [24–25]
and electrophoretically pure 15-LO from reticulocy tes
[26]. Using the biomembrane models consisting of
phospholipids (lecithin or phosphatidylinositol) and
linoleic acid, it has been shown that PUFAs oxidation
carried out by potato 5-LO proceeds directly on the
membrane [27–28]. The main factors providing the
enzyme sorption on the membrane surface are hydro-
phobic and electrostatic interactions. It was established
that with the replacement of certain amino acid resi-
dues in the 15-LO molecule from reticulocytes, the
hydrophobic bonds are formed between Phe70, Trp181,
Tyr15, Leu71 Leu195 and lipid part of the membrane;
these are tryptophan residues in the case of 5-LO from
leukocytes (Trp13, Trp75, Trp102) [24]. Isolated from hu-
man 5-LO, the PLAT domain was able to aggregate
and therefore could not be used to study the interac-
tions. A substitution of the membrane-binding tryp-
tophan 75 with glycine led to reducing the aggregation
and increased its thermal stability [21].
The LO sorption on the membrane surface causes
changes in the enzyme molecule, in particular, the
changes in the protein confi guration. Also, an addi-
tional regulatory effect on the LO activity infl uences
the biochemical and physicochemical properties of
the membrane matrix. According to [25], the 5-LO
molecule from leukocyte is located on the membrane
surface at approximately 450; one of tryptophans
(probably Trp75) is inserted into the hydrophobic
layer to a depth of 8-9 A0 from the membrane centre.
Trp13 and Trp102 interact with the surface but are not
inserted into the lipophilic membrane layer. This
collocation allows tight fi xation of the enzyme mol-
ecule and reduces the distance between the LO ac-
tive center and the membrane surface, where the
substrate reaction occurs. Similar processes can sig-
nifi cantly alter the enzyme conformation and its cat-
alytic properties. The interaction of the 13-LO from
soybeans with the surface of phosphatidylcholine
micelles changes the enzyme specifi city at the ca-
talysis of equimolar mixture of 13-hydroperoxide
and 9-hydroperoxide of linoleic acid [29].
Phospholipids
The composition of membrane structures is an im-
portant factor in the regulation of the enzyme sorp-
tion. The published data are contradictory when it
concerns the infl uence of phospholipids (PL) with
different charges on the LO activity (Table 1). For
instance, a high affi nity of the animal enzymes to
phosphatidylcholine was shown, in contrast to ani-
onic phospholipids [18, 24, 26, 35, 53]. During puri-
fi cation of 5-LO from human leukocytes, it was ob-
served that the enzyme activity depends on micro-
somal membranes [30] since the synthetic phos-
phatidylcholine (PC) vesicles, which are similar to
the cell membrane fraction, act as the enzyme stimu-
lating factor. PC promoted Ca2+ stimulation of the
5-LO activity in vitro [35]. In the presence of Ca2+,
the isolated C2-like β-sheet domain of 5-LO has a
higher affi nity to the PC zwitterion vesicles than to
the vesicles of anionic phosphatidylserine (PS) and
phosphatidylglycerol. The selectivity of 5-LO asso-
ciation with the nuclear membrane is determined by
the specifi city of the enzyme binding with PC (a nu-
clear membrane is rich in PC) and this association is
Ca2+-dependent [24–25]. Three tryptophans (Trp13,
Trp75, Trp102) were identifi ed, which participate in
the interactions of ligand-binding loop [2].
It is assumed that the 5-LO selectivity to PC is im-
portant for the enzyme contact with the nuclear en vi-
ronment [24] as β-sandwich is required for tran slo-
cation of soluble 5-LO from cytosol to the nuclear
mem brane [55]. It is believed that calcium ions pro-
mote the association of C2-like domain of 5-LO (as
well as phospholipase A2) with the PC membrane in
two ways: local neutralization of anionic surface of
the protein, and changing the orientation of ali pha tic
and aromatic side chains of amino acid residues in
the Ca2+-binding loop, which leads to their incorpo-
164
T. D. Skaterna, V. M. Kopich, G. I. Kharitonenko, O. V. Kharchenko
ration into the membrane and hydrophobic interac-
tion [24]. The Ca2+-induced interaction with PC sta-
bilizes 5-LO and the membrane, and results in the
enzyme activation [18].
The other results were obtained in the study on the
interaction of the potato tubers 5-LO with differently
charged phospholipids. For this enzyme, the activat-
ing effect of phosphatidic acid (PA) [48], phosphati-
dylinosite (PI) [27], phosphatidylserine (PS) [27] in
the micellar system was demonstrated.
The differences in the PL effects on the enzymes
from plant and animal sources can be explained com-
paring the affi nity of these enzymes to diversely cha-
rged phospholipids, which translocate to the mem-
Table 1. Lipid compounds and action on lipoxygenase enzymes
Lipid compounds Enzyme Action on enzyme Reference
PC vesicles 5-LO human leukocytes activation [30]
1-palmitoyl-2-arachidonoyl-sn-
glycero-3-phosphocholine
5-LO human activation of membrane binding [25]
sulfated derivatives of
galactocerebroside
5-LO human inhibition in the cell [31]
1-oleoyl-2-acetylglycerol 5-LO human stimulation by binding with C2-like domain [32]
cholesterol, cholesterol sulfate,
cholesterol phosphate
5-LO human inhibition of activity [33]
arachidonic acid 15(S)-HETE 5-LO human enzyme translocation to the membrane [34]
PC С2-like domain of 5-LO human affi nity to PC vesicles in Ca2+ presence [35]
NO-LA, NO-OA human PMNL 5-LO inhibition of activity [36]
5-HPETE, 12-HPETE,
15-HPETE, 13-HPODE
5-LO mammalian enzyme activation [37–38]
13-HODE 15-hLO-1 change of substrate specifi city [39]
15-HETE 15S-LO enzyme activation [40]
12-HETE 15-hLO-1 increase of substrate specifi city towards
AA over LA
[41]
oxo-lipids 15-LO human inhibition of activity [42]
(Z)-9-octadecenyl sulfate 15-LO human inhibition of activity [43]
13-HODE 15-hLO-2 epithelial changing the substrate specifi city [41]
13-HPODE 15-hLO-2 epithelial effect on substrate specifi city [44]
13-HPODE 15-LO-1 human reticulocyte effect on substrate specifi city [44]
oxo-lipids: 5-oxo-ETE, 15-oxo-
ETE, 12-oxo-ETE
12-LO human inhibition of activity [42]
13(S)-hydroxyoctadeca-
9(Z),11(E)-dienoic acid
12S-LO human platelete enzyme dimmerization [45]
PC, PI 12-LO porcine leukocytes inhibition of activity [46]
(Z)-9-octadecenyl sulfate LO-1 soybean inhibition of activity [43]
12-HETE LO-1 soybean increase of substrate specifi city [47]
PC vesicles 13-LO soybean changes of equimolar mixture products
of 13-HPODE/9-HPODE
[29]
PA 5-LO potato tubers enzyme activation [48–49]
PI 5-LO potato tubers enzyme activation [27]
PS 5-LO potato tubers enzyme activation [27]
LHA 5-LO potato tubers
12-LO porcine leukocytes
15-LO rabbit reticulocyte
13-LO soybean
inhibition of activity [50–52]
165
Lipoxygenase regulation in vivo and in vitro by lipid compounds
brane through the same mechanism [19, 24]. Affi nity
to a particular lipid is associated with the enzyme
localization in the cell. So, the affi nity of phospholi-
pase A2 and 5-LO from leukocytes to PC is deter-
mined by their location on the nuclear membrane
surface where the molar moiety of phospholipid is
48 %. As regards protein kinase C and 5-LO from
potato tubers, they demonstrate affi nity to anionic
PL, which is explained by the sorption of these en-
zymes on the plasmalemma surface enriched in ani-
onic PL. 1-palmitoyl-2-arachidonoyl-sn-glycero-3-
phosphocholine caused the enzyme connection and
deeper penetration. This may assist 5-LO to be clos-
er to the nuclear environment composed of lipids
with a high content of arachidonic acid [25].
The 5-LO from animal sources interacts also with
cationic phospholipids [18]. This interaction is stro-
nger and occurs in the absence of calcium ions al-
though they increase the enzyme activity. It was sug-
gested that 5-LO can bind to the membrane in «pro-
ductive» or «unproductive» manner although bind-
ing to the membrane surface does not activate 5-LO
itself. In turn, it was demonstrated that anionic sul-
fated derivatives of galactocerebroside (sulfatides
lipids) inhibit the 5-LO activity in the cell [31].
Monoglycerides and diacylglycerols
Monoglycerides and diacylglycerol are other types
of lipids that can activate 5-LO. The most effective
activating compounds are 1-oleoyl-2-acetylglycerol
(OAG), 1-O-hexadecyl-2-acetyl-sn-glycerol and
1,2-dioctanoyl-sn-glycerol [32]. Ca2+ prevents the sti-
mulating effect of OAG; there is no activating effect
of OAG in the presence of phospholipids or cell me-
m brane. The mutant 5-LO with three tryptophan re si-
dues (Trp13, – 75, – 102) in the C2-like domain was
not stimulated by OAG. It was established that these
residues are involved in the interaction of 5-LO with
OAG, and that gives us a reason to believe that OAG
directly stimulates 5-LO by the interaction with pho-
spholipid-binding site located in the C2-like domain.
Another compound, diacylglycerol (DAG), is also
required for the association of the enzyme with the
nuclear membrane via the C2-like domain. Perhaps
this mechanism is similar to the enzymes with C2
domains in their structure and was investigated for
the protein kinase C in more details. The protein ki-
nase Cε deeply penetrates to the plasma membrane
with C2 domain depending on the DAG generation
due to the activity of phospholipase D / phosphatidic
acid phosphatase [20]; the protein kinase C2 domain,
activated by Ca2+ in phosphatidylserine-dependent
way, binds to the membrane whereas the C1 domain
is involved in the following immersion into the
membrane and binding with DAG [18].
Uncharged glycerol seems to bind with the C2-
like domain without charge neutralization or changes
in the orientation of the enzyme side chain caused by
calcium ions. OAG, like Ca2+, protects 5-LO from
the glutathione peroxidase-1 inhibitor [32]. OAG is
a result of the phospholipase D activity in the cell.
Preincubation of human polymorphonuclear leuko-
cytes with the phospholipase D inhibitor resulted in
decreased synthesis of 5-LO products and blocked
the 5-LO translocation from cytosol to the nuclear
membrane [56], whereas OAG (30 μM) reversed the
inhibitory effect of 1-butanol on the synthesis of
5-LO products.
Cholesterol, cholesterol sulfate
The addition of cholesterol to the membrane prepa-
ration (20 %) reduces the enzyme activity by half
[25] and cholesterol sulfate inhibits 5-LO in intact
cells [33]. The fact that the leukotrienes production
in the cell is regulated by cholesterol sulfate suggests
the possibility of regulatory role of sulfotransferases/
sulfatases in the 5-LO products synthesis. Cholesterol
sulfate regulates the activity of serine proteases, es-
pecially proteinkinase C isoforms, phosphatidylino-
sitol-3-kinase, and chymotrypsin. Cholesterol pho-
sphate, a synthetic anionic cholesterol derivative,
acts as a more potent inhibitor of the leukotrienes
synthesis than cholesterol sulfate [33]. According to
the proposed mechanism of action, cholesterol and
its derivatives may inhibit the protein-lipid interac-
tions of the C2 domain of 5-LO enzyme with phos-
pholipase A, which interacts strongly with zwitterion
phosphatidylcholine. These interactions reduce the
substrate release from 5-LO and thus decrease the
enzyme activity in the cell. The cholesterol sulfate
166
T. D. Skaterna, V. M. Kopich, G. I. Kharitonenko, O. V. Kharchenko
structure is similar to tirucallic acid, which directly
binds to the 5-LO protein [57].
Polyunsaturated fatty acids (PUFAs)
and their derivatives
There are the data demonstrating LO regulation by
other lipids PUFAs. Interestingly, the 5-LO substrate
arachidonic acid (AA) can regulate the 5-LO translo-
cation in human neutrophils [34]. An application of the
redox and competitive 5-LO inhibitors and experi-
ments with the FLAP inhibitor and the intracellular
Ca2+ chelator demonstrated that the AA-re gu lated
5-LO translocation is FLAP- and Ca2+-dependent.
Altogether, the facts indicate the regulation of 5-LO
translocation by AA which exists at/or separately of
the catalytic site. Moreover, the oxidized de rivatives
also infl uence LO binding: 15(S)-HETE stro ngly in-
duces the 5-LO translocation whereas 12(S)-HETE
does not [34]. The specifi c LO products can al so acti-
vate some types of LO. Perhaps, AA can bind directly
with the enzyme molecule like it was desc ri bed for the
AA/protein kinase C [58]. AA was shown to activate
directly protein kinase C, involving a sequential C2.
This suggests a model of activation, in which an in-
crease in intracytosolic Ca2+ leads to the interaction of
arachidonic acid with the Ca2+-binding region; only
after this step, does the C1A subdomain interact with
arachidonic acid, leading to the complete activation of
the enzyme. Com parison of phospholipase A2 (which
has C2 and catalytic domains) and 15-LO [59], shows
that the association of the membrane surface promotes
the correct orientation of amino acid residues in the
catalytic domain of 5-LO. The oxidized PUFAs are
also able to infl uence the substrate specifi city as it was
described for 13-HODE, which can change the sub-
strate specifi city of 15-hLO-1 [47].
The products of one LO isozyme can be used to
activate another LO isozyme, potentially regulating
each other’s activity in the cell. It have been shown that
5-HPETE, 12-HPETE, 15-HPETE, and 13-HPODE
activated 5-hLO [37–38], 15-HETE activated 15S-LO,
and 13-HODE activated epithelial 15-hLO-2 [40–41].
13-HPODE activated epithelial 15-hLO-2 and retic-
ulocyte 15-hLO-1 [44]. 12-HETE increased the sub-
strate specifi city of sLO-1 towards AA, when chal-
lenged with an LA/AA mixture [39]. 12-HETE in cre-
a ses also the substrate specifi city towards AA over
LA for 15-hLO-1 [47]. One of the major 15-hLO-1
products, 13-(S)-hydroperoxy-9,11-(Z,E)-octadecadie-
noic acid (13-HPODE) from LA, upregulates the MAP
kinase signaling pathway, the major 15-hLO-2 product,
15-(S)-HPETE from AA, down-regulated MAP kinase.
The reaction of nitric oxide and nitrite-derived spe-
cies with PUFAs generated electrophilic fatty acid
nitroalkene derivatives with NO2: nitro-oleic (NO-
OA) or nitro-linoleic acid (NO-LA) caused the con-
centration-dependent and irreversible inhibition of
the 5-LO activity in human PMNL induced alkyla-
tion of enzyme Cys418. NO-FAs acted as a selective
inhibitor for 5-LO and did not affect the activity of
the platelet-type 12-LO (ALOX12) or 15-LO-1-
(ALOX15) in intact cells or recombinant protein
[36]. Only 5-LO possessed functionally relevant nu-
cleophilic amino acids within the catalytic center as
potentially sensitive to an electrophilic attack.
Another N-containing derivative of PUFAs is lino-
leyl hydroxamic acid (LHA). LHA was shown to be
an inhibitor for pt5-LO [50], soybean 15-LO [52, 60],
porcine leucocytes 12-LO [51], rabbit reticulocyte 15-
LO. Using the model system of mixed micelles with
constant molar ratio, it was found that LHA acted as a
noncompetitive inhibitor of pt5-LO. The LHA oxy-
dized derivatives exhibited the same inhibition effects
as nonoxidized linoleyl hydroxamic acid on potato tu-
ber 5-LO and porcine leucocyte 12-LO. The pt5-LO
interactions with PA led to oxidation of nonspecifi c
reaction substrate – LHA [61]. These results suggest
that the enzyme activity can be potentially regulated
by this modifi ed inhibitor at the cell level.
A high affi nity of lipids to lipoxygenase is of use
for the synthesis lipoxygenase inhibitors. Some syn-
thetic lipid derivatives inhibit the lipoxygenase cata-
lysis. 1-oxyl-2,2,6,6-tetramethylpiperidinyl esters of
octadecanoic and dodecanoic acids decrease the rate
of the linoleic acid and linoleyl alcohol oxidation in
the micellar system catalyzed by 5-LO [62–63]. The
inhibition mechanism is proposed, which includes
the interaction of lipophilic nitroxyl compounds with
the radical intermediate formed in the catalytic proc-
ess and the blocking of free radical transformation. It
167
Lipoxygenase regulation in vivo and in vitro by lipid compounds
is demonstrated that the inhibition effect of fatty acid
derivatives is determined by the substrate nature and
the presence of allosteric effector.
Oxo-lipids
Recent publications have raised an interest to one
more type of lipids – oxo-lipids, which are a large
family of oxidized human lipoxygenase products. The
electrophilic oxo-lipids, such as 15-oxo-5,8,11,13-
(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE) and 12-
oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-
ETE), are derived hydroperoxyeicosatetraenoic acid
and hydroperoxyoctadecadienoic acid in the macro-
phage and are produced by conversion of the LO
products. It was demonstrated that 15-oxo-ETE ex-
hibited the highest potency against h12-LOX as an
inhibitor. 12-oxo-ETE had comparable potency
against h15-LOX-1 and inhibited h12-LOX. These
data could indicate selective regulation of h12-LOX
by the h15-LOX activity [42].
2. Mechanisms of lipids infl uence
Physical and chemical properties
of membranes
An infl uence of the membranes structural compo-
nents on the LO activity depends on their physical
and chemical properties. The greatest effect on sorp-
tion process of LO has fl uidity of the membrane
structure determined by the number of 1,4-cis,cis-
pentadien fragments consisting of fatty acids [25]
and the membrane surface charge. For lipids of large
unilamellar vesicles with increasing concentrations
of cationic lipid 1,2-dymiristoil-glycero-3-etyl phos-
phocholine, the 5-LO activity enhances, but in the
presence of anionic lipid 1,2-dipalmitoyl-sn-glyce-
ro-3-phosphocholine, the activity decreased [18].
The data obtained showed that unsaturation of lipid
acyl chain is a key modulator of the 5-LO activity
for both zwitterionic and anionic lipid membranes
[25]. The infl uence of cholesterol and cholesterol
sulfate (natural substance) on the LO activity is tho-
ught to be associated with changes in the physical
properties of the membrane surface since these sub-
stances are known as agents increasing the mem-
brane rigidity. As a component of membrane, cho-
lesterol sulfate plays a stabilizing role preventing
osmotic lysis, supporting adhesion of cells. The elec-
tric charge of the membrane surface affects the ori-
entation of polar phosphatidylcholine main groups
in the membrane: when the membrane surface charge
is negative, the positively charged ends of the choline
group move together into the membrane due to elec-
trostatic attraction. The penetration of cholesterol
sulfate stabilizes the membrane vesicles and reduces
the lipid bilayer fl exibility, namely, reduces the cho-
lesterol membrane fl uidity. The penetration of cho-
lesterol residues reduces the permeability of mem-
branes and increases their orderliness [33]. Thus,
membrane fl uidity is a key modulator of the mem-
brane binding and activity of 5-LO [25]. Another li-
pid phosphatidic acid (PA) is able to separate into
microdomains or induce a negative membrane cur-
vature due to the charged small head groups located
close to the bilayer acyl chains, a high affi nity to biva-
lent cations and a tendency to the formation of inter-
molecular hydrogen bonds. These properties lead to
destabilization of the PA lipid bilayer [64]. The
length and unsaturation differences between subst-
rates are essential for regulation of the LO activity as
it was shown for the human epithelial 15-lipoxygen-
ase-2 (15-LOX-2) with 13-(S)-HODE which changed
the substrate specifi city for arachidonic acid (AA)
and (γ)-linolenic acid (GLA); it indicates that the al-
losteric structural changes in the active site discrimi-
nate between AA and GLA to achie ve opposite ki-
netics effects [65].
The additional infl uence of PL is described as the
change of thermodynamic parameters of lipoxygen-
ase thermoinactivation [66]. The rate constants and
activation energy of enzyme thermoinactivation we-
re shown to increase in the presence of PA. It was
suggested that hydrophobic forces play an essential
role in the interaction between 5-LO and phospha-
tidic acid that can induce certain conformational
changes of the enzyme molecule.
Allosteric interactions
It was reported that a number of natural and synthet-
ic compounds affect the lipoxygenase activity by al-
168
T. D. Skaterna, V. M. Kopich, G. I. Kharitonenko, O. V. Kharchenko
losteric mechanism [43, 67–70]. A regulatory center
exists in the enzyme molecule, which shows affi nity
to both substances: activator and linoleic acid as was
described for (R,S)-2-hydroxy-2-trifl uoromethyl-trans-
n-octadec-4-enoic acid (HTFOA), a powerful acti-
vator of pt5-LO [71]. The kinetic isotope effect stud-
ies demonstrated that unsaturated sulfonic acids are
able to inhibit the activity of 15-LO from reticulo-
cytes and soybean LO-1 in the interaction with al-
losteric site of these enzymes [68, 70]. Compounds
(Z)-9-octadecenyl sulfate and (Z)-9-palmitoyl sul-
fate were replaced with the PUFA molecules consid-
ering two-fold higher affi nity for regulatory site
compared with the reaction substrate – linoleic acid.
The affi nity rised with an increase of carbon chain
length and slightly depended on the effector charge.
It is believed that hydrophobic bonds play a key role
in the interaction with allosteric regulator center.
This is supported by research of the nordihydro-
guaiaretic acid action. This compound is an inhibitor
of soybean, human 12- and 15-LO [72]; in return hy-
drophobic derivatives increase LO catalysis through
allosteric mechanism for human 15-LO. Therefore,
it is thought that the lipophilicity moiety of effectors
can change the regulatory infl uence of an active com-
pound on the enzyme activity. The membranes con-
Fig. 1. The proposed scheme of relationship of lipid components and the enzyme lipoxygenase in plant and animal cells.
Plant cell. 9-LO and 13-LO are localized in either a soluble or an associated form with membrane (13-LO is connected with
plastide membrane). LO oxidation of PUFAs takes place on membrane surface. PLD, PLA1, PLA2 liberate C18:2, C18:3 or C16:3
from phospholipids. PC, PI, PS and PA as natural membrane compounds can bind with 9-LO and change activity of LO regulated
level of oxylipins. It is assumed that PA as allosteric activator can induce formation of 9-LO dimmers in plant cell like it was dem-
onstrated for animal LOs.
Animal cell. In the resting cell, 5-LO is localized in either cytosol or in compartment inside the nucleus [22]. On activation, 5-LO
translocates to the nuclear envelope rich with PC, where enzyme connects with FLAP. PLA liberates AA from phospholipids. FLAP
is thought to participate in transfer of AA to LO. Molecules of AA increase translocation of 5-LO as well as 15(S)-HETE. PC, PI, PA
and Chol since natural membrane compounds can bind with LOs and change activity of LO regulated level of LO metabolites. 5-LO
can be a part of lipids rafts [9]. 12(S)-LO (in the presence of 13(S)-HODE) and 5-LO have ability to dimmerization. Oxo-lipids and
NO-derivatives of PUFAs inhibit 15-LO and 12-LO in the cell.
LO – lipoxygenase; HP – hydroperoxide; PL – phospholipids; PA – phosphatidic acid; PC – phosphatidylcholine; PI – phosphati-
dylinosite; PS – phosphatidylserine; Chol –cholesterol; AA – arachidonic acid; LA – linoleic acid; PLD – phospholipase D; PLA1,
PLA2 – phospholipase A1 and A2 respectively
169
Lipoxygenase regulation in vivo and in vitro by lipid compounds
taining lipids with unsaturated hydrocarbon chains
have a signifi cant stimulatory effect on the 5-LO ac-
tivity [25]. An importance of lipophilicity of the LO
allosteric regulator is coordinated with primacy of
hydrophobic bonds in providing the enzyme sorp-
tion on the membrane surface and involving allos-
teric regulation of these processes.
The membrane phospholipids phosphatidylcho-
line (PC) and phosphatidylinosite (PI) caused almost
complete disappearance of the S-shaped curve of Vst
depending on the substrate concentration of LA in
micellar system in studies with highly purifi ed prep-
aration of 5-LO from potato tubers [73], and both
phospholipids replaced the substrate molecules in
the regulatory site of 5-LO. Both phospholipids (PC
and PI) in the micellar system were shown to inhibit
another lipoxygenase 12-LO from porcine leukocy-
tes [46]. These lipids are able to compete with the
sub strate reaction LA in one of the centers (alloster-
ic) and change the enzyme affi nity to the substrate:
PI decreases Ks and Kns, whereas PC causes the op-
posite effect. Anionogenic phospholipid phosphati-
dic acid (PA) demonstrated the activation of pt5-LO
and replaced the substrate molecules in allosteric si-
te, that decreased the non-enzymatic product level [74].
These data suggest the compensatory action of natu-
ral components of membrane in the LO catalysis.
The PLAT domain has an ability to participate in
allosteric relationships. It plays a role in membrane
affi nity, allostery and substrate specifi city. Removal
of the PLAT domain affects the degree of allostery
and moderates the communication pathway between
the allosteric and catalytic sites [59].
The proposed scheme of relationship of the lipid
components and the enzyme lipoxygenase in plant
and animal cells is presented in Figure 1.
Dimmerization of proteins
The recent data point to the LO capability to transit to
the dimmer state, which was demonstrated for the hu-
man 5-LO [75]. In aqueous solutions, the rabbit 12/15-
LO is mainly present as a hydrated monomer. The
rabbit 12/15LOX functions as a monomer that domi-
nates in solution, it dimmerizes at higher protein con-
centrations in the presence of salt and increasing de-
gree of freedom of the N-terminal PLAT domain [75].
The human platelet-type 12S-LOX is stable as a dim-
er, in contrast to h-5LOX and r-12/15LOX, which are
monomeric. The enzyme undergoes ligand-induced
dimmerization in aqueous solutions under the action
of allosteric effector 13(S)-hydroxyoctadeca-9(Z),11
(E)-dienoic acid [76]. In the presence of Ca2+, 5-LO
from rat basophilic leukemia (RBL-1) cells demon-
strated the non covalent, monomer-dimer interaction,
and both forms of the enzyme were present and only
the high molecular weight species were active [77].
The possibility of LO dimmerization can be an expla-
nation of the allosteric mechanism which is character-
istic of majority of LOs.
Conclusion
5-LO can catalyze two reactions: the oxidation of
AA and formation of leukotriene A4. It was estab-
lished that the human 5-LO can form dimmers and it
explains that one monomer catalyzes the formation
of 5-HPETE and transmits to the second monomer
to form leukotriene A4 [75]. The phenomenon of LO
dimmerization can explain the ability of 5-LO to
catalyze two reactions and an allosteric behavior of
lipoxygenases. The LOX tendency to form dimmers,
where two noncovalently linked enzyme molecules
might work in unison [45], is a basis to understand-
ing allosteric interactions. The dimmerization phe-
nomenon seems to give a ground for explanation of
the number of binding sites on the protein surface
and the PL ability to decrease or increase this number,
which leads to changing the LO cooperativity with
the substrate. Lipid nature of the compounds can in-
fl uence the allosteric properties of LOs changing the
enzyme activity and level of its specifi c products.
This leads to regulation of LO activity via infl uence
on the protein-lipid interactions of C2 domain with
the membrane, changes in the enzyme affi nity, the
LOs translocation from cytosole to the membrane
surface, allosteric mechanism, and increasing selec-
tivity towards the substrate type. The lipophilicity
rate of effectors can change the regulatory infl uence
of active compound on the enzyme activity. To ex-
plain the LO catalysis, it is necessary to consider the
enzyme microenvironment and infl uence on the
170
T. D. Skaterna, V. M. Kopich, G. I. Kharitonenko, O. V. Kharchenko
range of LO products as well as a conformational
state of the protein molecule. Lipid microenviron-
ment is essential for the LO activity. This should be
considered in the regulation of a level of bioactive
lipoxygenases products because of the possibility of
an inhibitor molecule to be converted by the enzyme
at contacting with PL in the cell.
Acknowledgement
The authors acknowledge Tetyana Odarich, MD for
helpful assistance.
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Регуляція ліпоксигеназ in vivo та in vitro
сполуками ліпідної природи
Т. Д. Скатерна, В. М. Копіч,
Г. І. Харитоненко, О. В. Харченко
Ліпоксигенази (ЛО) відомі як одні з ферментів перекисного
окислення ліпідів. Більшість ЛО є розчинними ферментами
та характеризуються аффіністю до мембран. Транслокація
ферменту з цитозолю на мембранну поверхню одна з стадій
регуляції рівня продуктів ліпоксигеназного каталізу у кліти-
ні. Сорбція на мембранну поверхню описана для більшості
173
Lipoxygenase regulation in vivo and in vitro by lipid compounds
ЛОз з рослинних та тваринних джерел. Даний огляд пред-
ставляє дані по регуляції ЛОз активності природніми та хі-
мічно модифікованими речовинами ліпідної природи. Здат-
ність ліпідів регулювати ЛО активність може здійснюватись
через: білок-ліпідні взаємодії C2 домену з мембраною, змі-
ну аффінності ферменту, транслокацію ЛОз, алостеричну
регуляцію, збільшення селективності до субстрату. Ре гу ля-
торний вплив активної сполуки на ферментативну актив-
ність залежить від рівня ліпофільності ефекторів. При по-
ясненні ліпоксигеназного каталізу необхідно враховувати
вплив мікрооточення ферменту на рівень ЛО.
Ключов і слова: ліпоксигеназа, алостерична регуляція,
фосфоліпіди, інгібування, активація
Регуляция липоксигеназ in vivo и in vitro
соединениями липидной природы
Т. Д. Скатерная, В. Н. Копич,
А. И. Харитоненко, О. В. Харченко
Липоксигеназы (ЛО) известны как одни из ферментов пере-
кисного окисления липидов. Большинство ЛО растворимые
ферменты и характеризуются аффиностью к мембранам.
Транслокация фермента из цитозоля на мембранную повер-
хность одна из стадий регуляции уровня продуктов липок-
сигеназного катализа в клетке. Сорбция на мембранную
поверхность описана для большинства ЛОз из раститель-
ных и животных источников. Данный обзор представляет
данные по регуляции ЛО активности природными и хими-
чески модифицированными соединениями липидной при-
роды. Возможность липидов регулировать ЛО активность
может осуществляться через: белок-липидные взаимодейс-
твия C2 домена с мембраной, изменение аффинности фер-
мента, транслокацию ЛОз, аллостерическую регуляцию,
селективность к типу субстрата. Регуляторное влияние ак-
тивного соединения на ферментативную активность зави-
сит от уровня липофильности еффекторов. При объяснении
липоксигеназного катализа необходимо учитывать влияние
микроокружения фермента на уровень ЛО продуктов.
Ключевые слова: липоксигеназа, аллостерическая ре-
гуляция, фосфолипиды, ингибирование, активация.
Received 02.06.2015
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