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From the Department of Genetics,
Microbiology and Toxicology
Stockholm University
Biosynthesis and physiological functions of
N-‐acyl amino acids
Dominik Paweł Waluk
Stockholm 2012
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Cover illustration:
© Dominik Waluk, Stockholm 2012
ISBN
Printed in Sweden by US-AB, Stockholms Universitet
Distributor: Stockholms University Library
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Dedicated to my family
Science, freedom, beauty, adventure: what more could you ask of life?
Aviation combined all the elements I loved
Charles Augustus Lindbergh
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ABSTRACT
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LIST OF PUBLICATIONS:
1. Waluk D. P., Schultz N. and Hunt M. C.
Identification of glycine N-acyltransferase-like 2 (GLYATL2) as a transferase
that produces N-acyl glycines in humans.
FASEB J. (2010) 24 (8): 2795-803
2. Waluk D. P., Sucharski F., Sipos L., Silberring J. and Hunt M. C.
Reversible lysine acetylation regulates the activity of human glycine N-
acyltransferase-like 2 (hGLAYTL2): Implications for production of glycine-
conjugated signalling molecules.
J. Biol. Chem. In press . Epub 2012 Mar 9. Doi: 10.1074/jbc.M112.347260
3. Waluk D. P., Tillander V., Schultz N. and Hunt M. C.
Molecular characterization of two members of the glycine N-acyltransferase
gene family in human: glycine N-acyltransferase-like 1 (GLYATL1) and
glycine N-acyltransferase-like 3 (GLYATL3).
Manuscript (2012)
4. Waluk D. P., Vielfort K., Derakhshan S., Aro H. and Hunt M. C.
N-acyl taurines trigger insulin secretion by increasing calcium flux in
pancreatic β-cells.
Submitted (2012)
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Biosynthesis and physiological functions of N-acyl amino acids
I Introduction 8
1. Fatty acid amides as signalling molecules 8
1.1. N-acyl glycines 10
1.2. N-acyl taurines 14
1.3. Other N-acyl amino acids 16
2. Physiological regulation of N-acyl glycines 17
2.1. Biosynthesis and metabolism of N-acyl glycines 17
2.2. Human Glycine N-acyltransferases 21
2.2.1. Genetic polymorphisms of glycine N-acyltransferases 23
2.3. Post-translational modification of proteins 24
2.3.1. Lysine acetylation of eukaryotic proteins 25
2.3.2. Effects of lysine acetylation 27
2.3.3. Physiological and pathophysiological aspects of
lysine acetylation/deacetylation 27
2.3.3.1. Lysine acetylation and metabolism 28
2.3.3.2. Lysine acetylation in neurodegenerative disorders 29
2.3.3.3. Acetylation on lysine residues and cell migration 29
3. Physiological importance of N-acyl taurines 29
3.1. Transient receptor potential (TRP) channels 30
3.1.1. TRP channels in the β-cell 31
3.1.1.1. Vanilloid receptor TRP channels (TRPV) in β-cells 31
3.2. Calcium free ions signalling in the β-cells 32
3.3. Insulin secretion 33
II Aims of the thesis 37
III Results and discussion 38
4. Characterization of human glycine N-acyltransferase -like 2 (hGLYATL2) 38
5. Human GLYATL2 is regulated by acetylation/deacetylation of
lysine 19 39
6. Molecular characterization of novel human glycine
N-acyltransferases – hGLYATL1 and hGLYATL3 40
7. Other results on human glycine N-acyltransferase family 41
7.1. Three-dimensional (3D) structures prediction of hGLYAT proteins 41
8. N-acyl taurines regulate insulin secretion in pancreatic β-cells 44
IV Future perspectives 46
V Popular science 47
VI Acknowledgements 48
VII References 50
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Abbreviations
AA - arachidonic acid
ADH - alcohol dehydrogenase
BAAT - bile acid-CoA:amino acid N-acyltransferase
CB1 - cannabinoid receptor type 1
CB2 - cannabinoid receptor type 2
CIRC - Ca2+-induced Ca2+ release
CNS - central nervous system
COXs - cyclooxygenases
ER - endoplasmic reticulum
FAAH - fatty acid amide hydrolase
GLYT2a - neurotransmitter transporter subtype 2a
GLYATL2 - glycine N-acyltransferase-like 2
HAT - histone acetyltransferase
HD - Huntington Disease
HDAC - histone deacetylase
LC/MS/MS - liquid chromatography combined with tandem mass spectrometry
LOXs - lipoxygenases
NAGly - N-arachidonoyl glycine
NATau - N-arachidonoyl taurine
NATs - N-acyl taurines
NO - nitric oxide
OLGly - N-oleoyl glycine
OLTau - N-oleoyl taurine
PAM - peptidylglycine α-amidating monooxygenase
PMCA - plasma membrane Ca2+ ATPases
PPARa - peroxisome proliferator-activated receptor alpha
PTMs - post-translational modifications
SNP – single nucleotide polymorphism
TRP - transient receptor potential
TRPV1 - transient receptor potential vanilloid type 1
TRPV4 - transient receptor potential vanilloid type 4
VDCC - voltage-dependent Ca2+ channels
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I Introduction
1. Fatty acid amides as signalling molecules
Fatty acid amides are a group of endogenous lipid mediators that are of growing
interest and regulate a variety of cellular physiological functions throughout the body.
They contain one fatty acid and one amino acid, or an amine, or might involve other
nitrogen containing compound (hydrozides, acid azides, nitriles, isocyanates) linked by
an amide bond. This group of compounds have been identified in biological systems
for over 50 years (1), including bacteria, marine organisms and mammals (2-4). Fatty
acid amides can be divided into several classes (see Fig. 1) A) the endocannabinoids,
which contains N-acyl ethanolamines, N-acyl dopamines and acyl esters of glycerol
coupled with fatty acids, B) N-acyl amino acids, which are comprised of N-acyl
glycines, N-acyl taurines and other N-acyl amino acids like serine, alanine, tyrosine,
and finally C) primary fatty acid amides, which includes palmitamide, oleamide and
arachidonamide, that have so far been detected in biological systems (5-7). Following
the discovery of fatty acid amides in many biological systems, research is now focused
on their functions.
9
Fig. 1. Classification of fatty acid amides. Fatty acid amides are a large family of structurally diverse
molecules found in humans and other organisms. They are classified into endocannabinoids, N-acyl
amino acids and primary fatty acid amides.
The endocannabinoid system, discovered in the 1990’s, is a lipid signalling
system that is involved in regulating numerous processes in the central nervous system
(CNS) and in peripheral organs (8), including the perception of pain, anxiety and fear,
body temperature, control of appetite, metabolism, and these signaling lipids also have
important anti-inflammatory properties (9-15) (for review see (16)). Endocannabinoids
exert their effects mainly through cannabinoid receptors (CB1 or/and CB2) (17-19).
Additionally, some of the endocannabinoids binds to the peroxisome proliferator-
activated receptor alpha (PPARa) (20) and act as ligands for transient receptor
potential (TRP) vanilloid type 1 (TRPV1) channels, (for reviews see (20-22)).
Endocannabinoids show a strong structural relationship to N-acyl amino acids,
suggesting that these latter compounds may regulate similar signalling pathways.
One might expect that the structurally related N-acyl amino acids could act as
ligands for the CB1 or/and CB2 receptors like endocannabinoids, but the latest research
10
shows that N-acyl amino acids are not activating cannabinoid receptors (22, 23),
although they play significant biological functions in many organisms. Recently,
approximately 50 novel endogenous N-acyl amino acids have been identified from
extracts of rat brain or bovine spinal cord using a targeted lipidomics approach (4), and
these includes N-acyl glycines, N-acyl taurines, N-acyl glutamic acids, N-acyl serines
and others (4, 23). However, the functions of N-acyl amino acids are still unclear but it
is hypothesized that these lipids are putative signalling molecules with a wide rang of
biological activities (for review see (16)).
Another group of structurally related compounds are the primary fatty acid
amides. These compounds have been isolated and characterized from luteal phase
plasma (5) and cerebrospinal fluid from cat, rat and human (24, 25). One of the
primary fatty acid amides, oleamide, regulates the sleep/wake cycle, blocks gap
junction communication in glial cells, normalizes memory processes, decreases body
temperature and locomotion activity, stimulates Ca2+ release, modulates depressant
drug receptors in the CNS, and allosterically activates the GABAA receptors and
specific serotonin receptor subtypes (25-27).
This work is focused on N-acyl amino acids, namely N-acyl glycines, and the
next section will be dedicated to this class of bioactive molecules.
1.1. N-acyl glycines
The first annotation of glycine conjugates was 168 years ago, when Wilhelm
Keller carried out an experiment on himself, by injecting thirty-two grains of pure
benzoic acid in syrup, and the next morning collecting his urine (28). The experiment
was repeated three times, and the results were published in the Provincial Medical and
Surgical Journal in 1842. The crystalline mass from his urine turned out to be pure
hippuric acid (benzoyl glycine). That was the first known reaction in the human body
describing the conjugation of benzoic acid with glycine, to produce hippuric acid.
The conjugation of xenobiotics and endogenous carboxylic acids, particularly
acyl-CoA molecules with chain lengths of C6:0 – C8:0, with amino acids such as
glycine, is one of the major metabolic pathways in Phase II liver detoxification as it
enhances the water solubility of these compounds, thereby promoting their excretion in
substances such as urine and bile (29). A large amount of the glycine conjugation of
11
short- and medium- chain carboxylic acids has been shown to occur by the
mitochondrial amino acid conjugating system of the liver (30).
Glycine conjugates of medium- and long-chain saturated and
unsaturated fatty acids have recently been detected in the CNS (4). N-arachidonoyl
glycine (NAGly) was one of the first N-acyl glycines to be identified in vivo in rat
brain, spinal cord, small intestine, kidney and skin, at concentrations of approx. 50-140
pmol/g of dry tissue weight (23). NAGly was recently identified as an endogenous
ligand for orphan G-protein coupled receptors: GPR18 (31), GPR72 (32), GPR92 (33).
Huang et al have reported antinociceptive and anti-inflammatory effects by NAGly in
animal models of pain (23). In mouse macrophage RAW cells, NAGly showed anti-
proliferative properties (34). Low concentrations (~1 µM) of this compound induce
proliferation, whereas at higher concentrations (~10 µM) of NAGly inhibits
proliferation of T lymphocytes in vitro (35), and reduce proliferation of the human
rectal carcinoma cell lines, as reported recently by Gustafsson et al (36). In pancreatic
β-cells, NAGly induces intracellular calcium mobilization and insulin release (37).
Moreover, in 2006 Wiles et al showed inhibition of the glycine transporter (GLYT2a)
through direct, noncompetitive interactions by NAGly (38).
Another N-acyl glycine that has been characterized is N-oleoyl glycine
(OLGly). Formation of OLGly has been detected in partially purified rat brain lipid
extracts using liquid chromatography combined with tandem mass spectrometry
(LC/MS/MS) (39). Other results show the presence of OLGly in skin, lung, spinal
cord, ovaries, kidney, liver, and spleen at concentrations from ~750 to ~150 pmol/gram
of dry tissue weight (39). This novel endogenous lipid can act as a precursor of
oleamide, a primary fatty acid amide (27, 40, 41). OLGly was shown to regulate body
temperature and locomotion in rats (41).
N-palmitoyl glycine (PAGly) is another example of an N-acyl glycine and has
been detected at high levels in rat skin, lung, spinal cord, kidney and liver, at
concentrations from 1612 to 388 pmol/g of dry tissue (39) and it has been
demonstrated that this endogenous lipid acts as a modulator of calcium influx and
nitric oxide production in sensory neurons (42). PAGly has been clinically tested on
Caucasian women and confirmed to be an effective anti-wrinkle agent and might play
an important role in skin aging (43).
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Additionally, other N-acyl glycines like N-stearoyl glycine (STRGly),
N-linoleoyl glycine (LINGly) and N-docosahexaenoyl glycine, have been detected
throughout the CNS and the body (39). STRGly has been found mostly in skin (~1600
pmol/gram of dry tissue weight), LINGly mostly detected in lung, skin, kidney, liver,
ovaries, small intestine (~400 pmol/g to ~90 pmol/gram of dry tissue weight), and
N-docosahexaenoyl glycine has been identified mostly in skin, lung, spinal cord,
kidney and liver (~200 pmol/g to ~100 pmol/g of dry tissue weight) (39). The chemical
structures of N-acyl glycines are shown in Fig. 2.
O
NH
OH
N-octanoyl glycine(N-capryloyl glycine)
A
O
NH
OH
N-decanoyl glycine(N-caproyl glycine)
B
C O
NH
OH
N-dodecanoyl glycine(N-lauroyl glycine)
DO
NH
OH
N-tetradecanoyl glycine(N-myristoyl glycine)
O
O
O
O
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E O
NH
OH
N-hexadecanoyl glycine(N-palmitoyl glycine)
F
NH
OH
O
N-9-hexadecenoyl glycine(N-palmitoleoyl glycine)
O
O
G
NH
OH
O
N-octadecanoyl glycine(N-stearoyl glycine)
H
NH
OH
O
N-9-octadecenoyl glycine(N-oleoyl glycine)
O
O
I
NH
OH
O
O
N-9,12-octadecadienoyl glycine(N-linoleoyl glycine)
14
J
NH
OH
O
O
N-5,8,11,14-eicosatetraenoyl glycine(N-arachidonoyl glycine)
Fig. 2. Chemical structures of N-acyl glycines. A) N-capryloyl glycine (C8:0-Gly). B) N-caproyl
glycine (C10:0-Gly). C) N-lauroyl glycine (C12:0-Gly). D) N-myristoyl glycine (C14:0-Gly). E)
N-palmitoyl glycine (C16:0-Gly). F) N-palmitoleoyl glycine (C16:1-Gly). G) N-stearoyl glycine
(C18:0-Gly). H) N-oleoyl glycine (C18:1-Gly). I) N-linoleoyl glycine (C18:2-Gly). J) N-arachidonoyl
glycine (C20:4-Gly).
1.2. N-acyl taurines
A wide diversity of endogenous carboxylic acids and xenobiotics are
conjugated with amino acids, before excretion in urine or bile. Conjugates of bile acid
and carboxylic acids with taurine or glycine have been extensively characterized, and
de novo production of N-acyl taurines has been found in mouse peroxisomes (44).
Acyl-CoA:amino acid N-acyltransferase-like 1 (ACNAT1) has been shown to
efficiently conjugate very long- chain and long-chain, saturated fatty acids to taurine
(Km – 11 µM, Vmax – 159.5 nmol/min/mg, for N-palmitoyl taurine production) (44).
These taurine-conjugated fatty acids were detected in the CNS and peripheral tissues
like testes, kidney and liver of mice (45-47). However, in human another protein called
bile acid-CoA:amino acid N-acyltransferase (BAAT) functions as a conjugating
enzyme, mainly acting on bile acids and using taurine or glycine as acceptor
molecules, but BAAT conjugates long-chain and very long-chain fatty acid to glycine,
(Km – 19.3 µM, Vmax - 233 nmol/min/mg, for N-arachidoyl glycine production) (48).
BAAT has been purified from several species, e.g. rat (49), bovine (50), domestic fowl
(51), fish (52), and human (53). Physiological functions of N-acyl taurines (NATs) are
still elusive. N-arachidonoyl taurine activates TRPV1 and TRPV4 calcium channels in
mouse kidney with an EC50 value of 28 and 21 µM respectively and has been shown
that fatty acid amide hydrolase (FAAH) regulates level of NATs in tissues (47). One of
the latest studies on NATs are focused on where different biosynthesis pathways for
15
N-acyl taurines productions and action may be operated, then following by Long et al
liver appears to contain a highly active biosynthetic pathway for polyunsaturated
NATs, very high accumulated amounts of N-linoleoyl taurine
(C18:2-Tau), N-arachidonoyl taurine (C20:4-Tau) and N-docosahexaenoyl taurine
(C22:6-Tau) have been detected in mice liver and plasma treated with FAAH inhibitor
(PF-3845), however how these highly accumulated polyunsaturated N-acyl taurines are
released from liver and taken up into blood and other tissues remains unknown (54).
Interestingly, the same group showed that mice brain has slow process for production
long-chain saturated NATs, by detecting high accumulation of N-docosanoyl-taurine
(C22:0-Tau) was detected (54). Moreover our group has recently reported that PC-3
cells response in significant reduction of proliferation to treatment with N-arachidonoyl
taurine and N-oleoyl taurine even at concentrations as low as 1 µM (55). Recently, has
been shown that oxidative metabolism of N-arachidonoyl taurine in murine resident
peritoneal macrophages (RPMs) occurs by the action of lipoxygenases (LOXs) to
produce mainly 12-hydroperoxy eicosatetraenoic acid (12-HETE) (47, 56).
Metabolism of N-arachidonoyl taurine by LOXs may represent a pathway for
generation of bioactive signalling molecules. However, thou N-acyl taurines have been
extensively describe in mammalian systems like mice, still stayed undetected in human
and the pathway that produce these describes above and other subsets of NATs remain
poorly characterized.
The chemical structures of N-acyl taurines are shown in Fig. 3
A
NH
O
N-9-octadecenoyl taurine(N-oleoyl taurine)
B
NH
O
S
O
O
S
O
O
OH
OH
N-9,12-octadecadienoyl taurine(N-linoleoyl taurine)
16
NH
O
N-5,8,11,14-eicosatetraenoyl taurine(N-arachidonoyl taurine)
S
O
O
OH
S
N
O
O
O
OH
N-4,7,10,13,16,19-docosahexaenoyl taurine(N-cervonoyl taurine)
D
C
Fig. 3. Chemical structures of N-acyl taurines. A) N-oleoyl taurine (C18:1-Tau). B) N-linoleoyl taurine
(C18:2-Tau). C) N-arachidonoyl taurine (C20:4-Tau). D) N-cervonoyl taurine (C22:6-Tau).
1.3. Other N-acyl amino acids
As stated previously, approximately 50 novel endogenous N-acyl amino acids
have been identified in rat brain and bovine spinal cord using targeted lipidomics (4),
although function for many of these N-acyl amino acids have not been identified or
remain unclear.
N-acyl serines (e.g. N-arachidonoyl-L-serine) have been shown to regulate
homeostasis of the vascular system in addition to act as a vascular protective agent,
and acting as a relaxing factor in isolated mesenteric arteries in rats, which indicates
vasodilatory properties (57). N-arachidonoyl-L-serine was isolated from bovine brain
by Milman et at and reported that on rat isolated mesenteric arteries (EC50, 550 nM)
and abdominal aorta (EC50, ≈ 1,200 nM) plays as relaxation agent (57). This effect is
alike to the one caused by cannabidiol, a synthetic agonist of a novel cannabinoid- type
17
receptor (58). N-arachidonoyl-L-serine also suppresses formation of reactive oxygen
intermediates, nitric oxide (NO).
N-arachidonoyl alanine, an example of other N-acyl amino acid, has been
isolated from brain tissue from rats together with N-arachidonoyl glycine, (23)
however did not produce anti-nociception in a model of inflammatory pain in rat (59)
and N-arachidonoyl alanine is a very weak inhibitor of FAAH (60). However other
N-acyl alanines: N-linoleoyl-D-alanine together with a different N-amino acid linoleoyl
conjugates have recently been studied by Burstein et al, where they have showed that
in mouse macrophage RAW cells treatment with N-linoleoyl-D-alanine highly
increased production of PGJ (15-deoxy-Δ-PGJ2 – a prostaglandin D2 metabolite) (61),
which is reported in literature as an anti-inflammatory agent (62). It has been shown
that N-acyl alanines are active in the assays of RAW 264.7 cell function and a subset
of the in vivo assays of inflammation (63) however, the mechanisms underlying these
effects are unknown.
2. Physiological regulation of N-acyl glycines
N-acyl glycines have been proposed to be intermediary products in the
biosynthesis of primary fatty acid amides (40) but recent evidence suggests that they
exert physiological functions of their own. However there is still known so little about
their physiological effects throughout the body and furthermore, how these signalling
molecules are produced and how their biosynthesis is regulated currently is unknown.
2.1. Biosynthesis and metabolism of N-acyl glycines
Although N-acyl glycines have been identified in several tissues, the molecular
pathways for production and regulation of N-arachidonoyl glycine and other glycine
conjugates of fatty acids (N-acyl glycines) are not well understood. Three pathways
have been suggested for the formation of N-acyl glycines: 1) enzymatic synthesis by
glycine N-acyltransferases (GLYATs) (Paper I), 2) in vitro synthesis via cytochrome c
(42, 64, 65) and 3) in vitro oxidation by ADH7 (66). These three pathways are
described below and are shown in Fig. 4 and Fig. 5.
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In order to produce N-arachidonoyl glycine or the endocannabinoid
anandamide (arachidonoyl ethanolamine), free arachidonic acid (AA) is required. AA
can be generated by the action of lipases (phospholipase D, diacylglyceride) on
phospholipids. Once free AA is formed in cells, there are several possible pathways for
production of signaling lipids (containing AA in their structure) such as
N-arachidonoyl glycine (see Fig. 2.) or anandamide. Several results have already
demonstrated that anandamide can be synthesized enzymatically in rat and bovine (19,
67-70), either when several areas of brain like hippocampus, thalamus, cortex, ad
striatum, cerebellum, pons and medulla, or separate subcellular brain fractions like
synaptic vesicles, myelin, microsomal and synaptosomal membranes, were incubated
with free AA and ethanolamine. Anandamide can be selectively oxygenated by
lipoxygenases and cyclooxygenases (35) or hydrolyzed by fatty acid amide hydrolase
(FAAH) to arachidonic acid and ethanolamine (71). Furthermore, anandamide can act
as a precursor for NAGly. Anandamide has been shown to be a substrate for human
alcohol dehydrogenase (ADH7 isoenzyme) and it can be metabolized to
N-arachidonoyl glycinal, a newly identified intermediate, which is then converted by
aldehyde dehydrogenase to N-arachidonoyl glycine (see Fig. 5) (72).
19
Fig. 4. Pathways for production of signaling molecules from free arachidonic acid (AA). Free AA
is generated from the action of various lipases and can be metabolized in different metabolic pathways
(described in the text). FAAH – fatty acid amide hydrolase, HETE – hydroperoxy eicosatetraenoic acid,
COX-2 – cyclooxygenase-2, GLYATL2 – glycine N-acyltransferase-like 2, which is the focus of this
thesis is shown in purple.
It has recently been reported that cytochrome c can act on arachidonoyl-CoA
and glycine in the presence of hydrogen peroxide, which leads to the production of
NAGly (64). The same pathway is suggested for the enzymatic formation of N-oleoyl
glycine (65). Level of NAGly in vivo can be regulated by the action of FAAH, which
hydrolyzes NAGly to free arachidonic acid and glycine (73). NAGly is also a substrate
for selective oxygenation via cyclooxygenase-2 (74), or finally NAGly can be oxidated
by peptidylglycine α-amidating monooxygenase (α-AE) to the primary fatty acid
amide, arachidonamide (40).
Up to now the identification of an enzyme producing long-chain N-acyl
glycines has been elusive. Bile acid-CoA:amino acid N-acyltransferase (BAAT) has
been characterized as an enzyme conjugating acyl-CoA esters to taurine or glycine
(48), although at about 20% of its bile acid conjugating activity and BAAT conjugates
long-chain saturated acyl-CoA esters (from C16 carbons up to C20 carbons) to glycine
or taurine. Recently we showed that a previously uncharacterized acyltransferase in
human called glycine N-acyltransferase-like 2 (GLYATL2) is involved in glycine
conjugation of medium- and long-chain saturated and unsaturated acyl-CoA esters
(Paper I).
20
FREE ARACHIDONIC ACID
OH
O
NH
OHO
ANANDAMIDE
NH
OH
O
O
ARACHIDONOYL GLYCINE
H2N
OH
NH2
O
ARACHIDONAMIDE
ETHANOLAMINE
CoA
O
H2NOH
O
ARACHIDONOYL-CoA
GLYCINE
GLYATL2
ALCOHOL DEHYDROGENASE(ADH7)
ARACHIDONOYL GLYCINAL
NH
HO
O
ALDEHYDE DEHYDROGENASE
FAAH
FAAH
H2NOH
ETHANOLAMINE
H2NOH
O
GLYCINE
H2NOH
O
GLYCINE
H2O2
cytochrome c
PEPTIDYLGLYCINE!-AMIDATING
MONOOXYGENASE
ARACHIDONOYL-!-HYDROXYGLYCINE
NH
OHO
O
OH
HOH
O
O
GLYOXYLIC ACID
Fig. 5. Biosynthesis and metabolism of N-arachidonoyl glycine. Free arachidonic acid (AA) can
be enzymatically converted to anandamide, and further metabolized to NAGly by the action of
ADH7 and aldehyde dehydrogenase. Free AA can also be esterified to its CoA ester by acyl-‐CoA
synthetases and the arachidonoyl-‐CoA can be a substrate either for hGLYATL2 (Paper I) or
cytochrome c to produce NAGly. NAGly can be metabolized by PAM (peptidyl-‐glycine α-‐amidating
mono-‐oxygenase) to produce arachidonamide.
21
2.2. Human Glycine N-acyltransferases
Glycine N-acyltransferase (GLYAT) is well known to be involved in the
detoxification of endogenous and exogenous xenobiotic acyl-CoA’s and in the
metabolism of fatty acids in mammals (for review see (75)). GLYAT is an amidating
enzyme and as the name suggests, it conjugates the acyl-CoA esters of carboxylic acids
with glycine prior to their excretion in urine. GLYAT was first reported in the
literature as ‘glycine N-acylase’ (29), following its purification and characterization
from bovine liver mitochondria in the middle of the last century. The enzyme is said to
be liver and kidney specific, and has been purified from bovine, monkey and human
and has been characterized as a benzoyltransferase and phenylacetyltransferase in
mitochondria (30, 76), although conjugation of other aliphatic (C4:0 – C10:0) and
branched (iso- C4:0 – C6:0), as well as aromatic carboxylic acids (p-aminobenzoic, p-
hydroxybenzoic, cinnamic, α-picolinic), to glycine in the presence of GLYAT have
been reported (29). GLYAT is mainly a glycine conjugating enzyme, although it has
been shown to conjugate benzoyl-CoA with several other amino acids like alanine,
glutamic acid or serine, although the level at which these conjugates are formed is
relatively low, compared to glycine conjugation (77). It has been also reported that
GLYAT enzymatic activity might be age dependent, increasing until age eighteen and
later remaining constant until age forty (78). Recently, based on protein modelling
results, catalytic mechanism in which glutamic acid at position 226 in sequence (E226)
plays a key role in enzyme function has been shown (79). Enzyme activity results from
purified liver bovine and recombinant GLYAT have shown that this residue E226 is
required for full active enzyme and suggested to deprotonate glycine, which may allow
nucleophilic attack on the acyl-CoA.
Bioinformatics have identified a gene cluster of GLYATs in human and mouse
and these genes products show approximately from 20 up to 48% sequence identity to
each other in human (see Table 1).
hGLYATL1 hGLYATL2 hGLYATL3 hGLYAT 38% 40% 20%
hGLYATL1 - 48% 22% hGLYATL2 - - 21%
Table 1. Amno acid sequences identity between human glycine N-acyltransferases.
22
Comparison of sequences identity in human GLYAT proteins, suggests that they have
different substrate specificities and functions. These genes are localized on human
chromosome 11 position 11q12.1 (GLYAT, GLYATL1, GLYATL2), whereas GLYATL3
is situated on human chromosome 6 position 6p12.3 (see Fig. 6). All four gene
products are encoded by five exons. GLYAT and GLYATL1 (unpublished results) are
expressed in liver and kidney (Paper III); GLYATL3 in liver and pancreas (Paper III)
and GLYATL2 is mainly expressed in salivary gland and trachea, and also in brain
(Paper I).
GLYAT GLYATL2 GLYATL1 GLYATL3
5 Exons 5 Exons5 Exons 5 Exons
Fig. 6. Glycine N-acyltransferase genes on human chromosome 11q12.1 and chromosome 6p12.3.
Each gene is encoded by 5 exons. The gene products show approx. 20 – 48% sequence identity to each
other.
Merker et al speculated that GLYAT may be involved in the production of N-
acyl glycines (80). As these N-acyl glycines are now being recognized as bioactive
signaling molecules and as an enzymatic pathway for production of these has been
elusive, we therefore set out to examine if any of the uncharacterized GLYAT gene
products could be involved in production of long-chain N-acyl glycines. The enzymatic
reaction catalyzed by glycine N-acyltransferases is shown in Fig. 7
23
S
O
CoA
Oleoyl-CoA
+ H2NOH
O
Glycine
NH
OH
O
O
+ HS CoA
N-Oleoyl glycine
GLYATL2 glycine N- acyltransferase like 2
E.C.2.3.1.13
Fig. 7. Enzymatic reaction catalyzed by glycine N-acyltransferase-like 2. An example of the
reaction of human GLYATL2 with oleoyl-CoA in the presence of glycine, resulting in the production of
N-oleoyl glycine and free CoASH.
2.2.1. Genetic polymorphisms of glycine N-acyltransferases
DNA sequence variations that occur when a single nucleotide in the genome
sequence or other shared sequence is altered may be simply defined as a single
nucleotide polymorphism (SNP)
Five single nucleotide polymorphisms (SNPs), with three causing amino acid
changes in the protein of GLYAT have been identified in Japanese individuals, which
might affect the activity of this enzyme (81). Whether or not these SNPs affect the
GLYAT enzyme function need to be investigated. In compare to Japanese population
study on GLYAT, polymorphism within glycine N-acyltransferase in French
Caucasian population has been reported. Seven different polymorphysms of the
GLYAT gene within three missense mutations (S17T), (N156S), (R199C) were
described (82). Further bioinformatics analysis suggested that serine 17 (S17) is a
24
highly conserved amino acid among GLYAT orthologous proteins. Moreover, arginine
199 in the GLYAT protein sequence, based on bioinformatics, is thought to be also a
highly conserved amino acid, therefore substitutions to threonine (T) at position 17 and
to cysteine (C) at position 199 may have an influence on enzyme activity (82).
Glycine conjugation plays a major role in the metabolism and the detoxification
of various xenobiotics. Glycine N-acyltransferase (GLAYT) is a conjugating/amidating
enzyme, and it thought to conjugate the acyl-CoA ester of carboxylic acids with
glycine prior to their excretion in the urine (83-87). Genetics variation of the GLYAT
gene may be involved in therapeutic response to xenobiotics that are metabolite and
conjugated to glycine or other amino acids in Phase II liver detoxification pathways.
There are yet no reports on SNPs in other GLYATs. However we can
hypothesise that single nucleotide polymorphism may play an important role in
regulation of GLYAT proteins functions. Therefore extended study in this research
area need to be continued, which would lead us to better understanding nature of
signalling molecules – N-acyl amino acids, where contribution to their production by
glycine
N-acyltransferase proteins is beyond doubt.
2.3. Post-translational modification of proteins
Proteins can be subject to a large number of post-translational modifications
(PTM). Over 200 different PTMs have been described such as glycosylation,
acetylation, alkylation, methylation, glycylation, biotinylation, glutamylation and
ubiquitination. The PTMs allow a large extension of the protein repertoire and also
make it possible to regulate proteins/enzymes without the need for de novo protein
synthesis. PTMs play an important and crucial role in modifying the end product of
gene expression and contribute towards biological processes and disease conditions
(for review see (88)). PTMs also help in utilizing identical proteins for different
cellular functions in different cell types. Cross-talk between different PTMs increases
the complexity of regulatory signals substantially.
Lysine residues can be acetylated, ubiquitinated, sumoylated, neddylated or
methylated (for reviews see (89, 90)). The PTMs on lysine are mutually exclusive and
allow extensive cross-talk between signalling pathways, which can underlie regulatory
25
programmes in development or pathogenesis. For example, phosphorylations are often
the first response to extracellular stimuli and the signals are subsequently converted to
acetylation-based responses (89). Another example is p53 acetylation, which can
compete with ubiquitination and thus influence the stability and half-life of p53 (89,
91, 92). Moreover, latest data suggest that p53 acetylation at K373, K381 and K382 is
essential together with phosphorylated (at residue T146) H1.2 – a linker histone to
serve as signals to command p53-regualted transcriptional program in response to
DNA damage (93), means that not only acetylation of p53 but even cross-talk with
phosphorylation on another protein (H1.2) plays major function in p53 activation.
2.3.1. Lysine acetylation of eukaryotic proteins
Acetylation is the most common covalent modification out of the several
hundred that have been reported to occur on eukaryotic proteins. Acetylation and
deacetylation of proteins, especially histone tails, have been studied since 1968 (92).
Acetylation on the N-terminal site of proteins is irreversible and occurs on the bulk of
eukaryotic proteins, while the e-amino group on lysine residues can be reversibly
acetylated. Until recently lysine acetylation has been known to be common on
histones, transcription factors, nuclear receptors and α- tubulin (94). In this
introduction, I would like to focus mainly on the emerging role of lysine acetylation of
non-nuclear proteins.
NH3N COO
OH
H2NCH3
SCoAO
CoASNH3N COO
OH
HN CH3
O
HATsHDACs
H3C O
O
OH
26
Fig. 8 Acetylation of proteins is catalyzed by a wide range of acetyltransferases that transfer acetyl
groups from acetyl-coenzyme A to either the a-amino-terminal residue or to the e-amino group of lysine
residues of proteins at various positions. Acetyl-CoA dependent lysine ε-NH2 group acetylation.
HATs – histone acetyltransferases, HDACs – histone deacetylases.
The first histone acetyltransferase enzyme was identified in the middle of the
1990s, and linked histone acetylation to gene regulation (92, 95). There are three major
families of HATs: 1) general control non-derepressible 5 (Gcn5)-related N-
acetyltransferases called GNATs, 2) p300/CBP (CBP stands for CREB binding
protein), 3) MYST (Moz, Ybf2/Sas3, Sas2, Tip60) proteins (96-98).
One of the first proteins identified as a substrate for deacetylating enzymes
were histones (92). Enzymes responsible for this conversion belong to a family called
histone deacetylases (HDAC). This family of deacetylases is divided into two groups,
one contains 11 members and might be called ‘classical’, which means they require
Zn2+ to efficiently promote protein deacetylation and work as simple hydrolases, by
releasing the acetyl moiety as acetate (see Fig. 8). The second group includes 7
members called sirtuins or sirt, where sirtuins stands for (Silent Information Regulator
Two (Sir2) proteins) (99, 100). These enzymes work as nicotinamide adenine
dinucleotide (NAD) - dependent histone deacetylases (92, 99, 101). The mechanism of
deacetylation by sitruins is shown in Fig. 9.
Fig. 9. The stoichiometry of the reaction of NAD-dependent sirtuins in the deacetylation of
ε-acetyl-lysine residues. The acetyl group bonded to lysine is marked in red.
N-Ac-Lysine – N-acetylated lysine residue.
27
2.3.2. Effect on lysine acetylation
The effects of lysine acetylation on proteins can result in neutralizing a positive
charge, adding bulk, increasing hydrophobicity or impairing the ability to form
hydrogen bonds. Acetylated lysines are more likely to be found in helices and less
likely in coils. Acetylation may modulate protein-protein, protein-ligand, protein-DNA
interaction, protein stability or intracellular localization (94) and play a crucial role in
many physiological processes such as migration, metabolism and ageing as well as in
pathological diseases such as cancer and neurodegenerative disorders (for review see
(101)). This strongly suggests that the biological processes that involve protein
acetylation and their physiological relevance are mostly likely severely
underestimated.
The acetylation effects can be classified into three groups. A) Single residue
acetylation may act as a simple on/off switch, for example the inactivation of acetyl-
CoA synthetase-2 by acetylation (102) and acetylation of a-tubulin, to activate cargo
transport on microtubules (89, 103), B) Acetylation may cover charged patches, where
it is the number of acetylated residues that count and not which particular residue in
the charged patch that is modified. An example of this mechanism is found in cortactin
(which regulates cell motility), which is acetylated on about 10 lysine residues in a
repeat domain (104). Each repeat has at least one lysine and each repeat is sufficient
for F-actin binding. Finally, C) acetylation may result in a site-specific effect that
influences the affinity for interaction partners; an example of this is ornithine
carbamoyltransferase, whose activity for one of its substrates, carbamoyl phosphate, is
regulated by acetylation of K88 in the enzyme (105).
2.3.3. Physiological and pathophysiological aspects of lysine
acetylation/deacetylation
Lysine acetylation is a major post-translational modification of proteins. This
highly conserved from prokaryotes to eukaryotes form of proteins modification
regulates many physiological processes such as metabolism, cell migration, ageing and
inflammation. Acetylation/deacetylation is catalyzed by acetyltransferases and
deacetyltransferases respectively and deregulation of this greatly controlled enzymatic
28
process may lead pathological diseases such as diabetes, obesity, cancer and
neurodegenerative disorders. This work is focused on lysine acetylation/deacetylation
of metabolic proteins and some of major aspects in this matter are presented below.
2.3.3.1. Lysine acetylation and metabolism
Lysine acetylation can control the activity of metabolic enzymes.
Mitochondrial acetyl-CoA synthetase activity, for instance, is controlled by reversible
acetylation of lysine 642 (K642) in the enzyme’s active site (102). The enzyme is
activated by the soluble mitochondrial sirtuin SIRT3, which deacetylates it.
Hyperacetylation of proteins has been found in SIRT3 knockout mice and this
indicates multiple substrates for SIRT3 (106). Several homologues of Saccharomyces
cerevisiae Sir2 are located in the mitochondria, which implies that even more lysine-
acetylated sirtuin substrates exist in this organelle. Using proteomic analysis, Kim et al
have found that more than 20% of mitochondrial proteins are acetylated in mouse liver
and this might play an important role in regulating energy metabolism, including six
tricarboxylic acid (TCA) cycle proteins, 26 proteins involved in oxidative
phosphorylation, 27 β-oxidation or lipid metabolism proteins, transporter and channel
proteins and several other transporters (107). All these discoveries are strong support
that acetylation of proteins may influence the mode or rate of energy production or
other mitochondrial functions.
Pancreas regulates glucose homeostasis by regulating insulin secretion in
response to continuous changes in glucose concentration in blood. It has been shown
that lipotoxicity induce β-cells apoptosis and dysfunction in pancreatic islets (108).
Recent years research show that lysine deacetylases (HDACs) inhibitors promote
development, proliferation and differentiation in animal models of diabetes and insulin
resistance (108-110). Further, over expression of SIRT1 in rat pancreatic β-cells,
inhibits NFκB activation, therefore SIRT1 protects β-cells cells from cytokine-induced
toxicity (111, 112). Summing up, latest research links pancreas function with activity
of lysine deacetylases all classes (HDAC and Sirtuins), which may suggest therapeutic
roles in preventing lipotoxicity-induced apoptosis and dysfunction in the pancreas by
controlled level of deacetylases activity, which may be accomplished by lysine
deacetylases modulators (for review see (113)).
29
2.3.3.2. Lysine acetylation in neurodegenerative disorders
The importance of acetylation in pathogenesis has recently been shown in
Huntington disease (HD), which is an incurable neurodegenerative disease caused by
neuronal accumulation of mutant protein Huntingtin (Htt) (114, 115). Mutant Htt
contains characteristic repeat sequences of glutamines near the N-terminus site. The
number of repeats in Huntingtin increases with time in pathogenic mutants. This can
cause protein aggregation in primary striatal and cortical neurons and results in
neuronal degeneration. Degradation of mutant Htt is an acetylation-dependent process
and it has been found that acetylation on residue K444 targets mutant Huntingtin into
autophagosomes, where the mutant protein is degraded (116). This clearance
mechanism can reverse the neurotoxic effect of mutant Htt protein. In mutant
Huntingtin, when residue K444 cannot be acetylated, the mutated Htt been shown to
accumulate in mouse brain and cultured neurons (116).
2.3.3.3. Acetylation on lysine residues and cell migration
Lysine deacetylation can control cell migration by controlling microtubule-
dependent cell motility with over-expression of HDAC6 in NIH3T3 cell line (117).
Other studies show that the actin cytoskeleton is the main target of HDAC6 activity for
the regulation of cell migration, at least in fibroblasts (118, 119). All these studies
confirm the critical role of the actin cytoskeleton in the link between protein
acetylation and cell migration.
3. Physiological importance of N-acyl taurines
Taurine is a bioactive amino acid highly abundant in the brain and taurine
conjugates is mostly characterized in bile acid synthesis, where taurine-conjugated bile
acids are well established metabolites in the liver (120). N-acyl taruines (NATs), as it
was mentioned before in chapter 1.2., were recently discovered in mass spectrometry
analysis of wild-type and FAAH knockout mice (45, 46). Mice that were lacking the
FAAH enzyme showed in brain and spinal cord significant levels of saturated and
30
monounsaturated very long-chain NATs. The long-chain unsaturated N-acyl taurines
like N-arachidonoyl taurine (NATau) and N-oleoyl taurine (OLTau) have been found
in liver and kidney, however their levels were not considerably elevated in the FAAH
deficient mice. As it was mentioned before N-arachidonoyl taurine was shown to
activate TRPV1 and TRPV4 channels and reported to be a ligand for GPR72.
All together, gathered information point to the cell-signalling role for these
taurine-conjugated lipids and high potential of other yet undiscovered purpose for
these signalling molecules. This study has also been addressed to new physiological
functions of N-acyl taurines (Paper IV).
3.1. Transient receptor potential (TRP) channels
The transient receptor potential (TRP) channels were discovered and first
described in the photoreceptor cells of blind fruit flies (121, 122). It has been passed
three decades (first report from 1982) from that discovery, and up to date there are 28
TRP channels characterized, within 27 are expressed in human. TRP channels are
present in almost every each type of cells and they have a variety of regulatory
pathways and physiological functions, including detection of various physical and
chemical stimuli in vision, taste, olfaction, hearing, touch, pain and temperature,
pheromones, osmolarity (123). TRP channels are tetrameric ion channels and may
form homo as well as heterotetramers, which give them opportunity to form many
variations of channels. TRP channels have been divided into two groups with seven
subfamilies. Group one consists of five subfamilies: TRPC (C for canonical) with
seven members, there are six channels related to the vanilloid receptor (TRPV), eight
related to the melastatin subfamily (TRPM), each of TRPA subfamily members
(TRPA, TRPA1) contain many ankyrin repeats at N terminal site and last subfamily is
called no mechanoreceptor potential C TRP channels (TRPN) ((124), for review see
(125)). Group two have 2 subfamilies, three mucolipin (TRPM) channels and three
polycystin channels (TRPP).
Thermosensitive TRP channels usually play role as multimodal receptors with
respond to various chemical and physical stimuli. Nine thermosensitive TRP channels
have been identified in mammals to date (for review see (126)) . TRPV, TRPM and
TRPA subfamilies channels are activated by physiological temperature for each
31
species. TRPV1 and TRPV2 are activated by raised temperature (> 42 °C), by warm
temperature TRPV3, TRPV4, TRPM2, TRPM4 and TRPM5 are activated, whereas
cold and cool temperatures stimulate TRPM8 and TRPA1. At this time, it is clear,
there is no unifying theme in TRP proteins function or mechanism for activation
3.1.1. TRP channels in β-cells
For the past three decades, from discovery of TRP channels, cellular expression
several of them have been reported in pancreatic β-cells (127, 128): TRC1-6 (112,
129), TRPM2-5 (130-134) and TRPV1, TRPV2, TRPV4, TRPV5 (135-138). It has
been shown that several members of TRP channels expressed within the islets of
Langerhans (139) mediate insulin secretion from pancreatic β-cell lines. Other
members of the TRP family of cation channels known to function in regulation of
insulin secretion are expressed in mouse, rat or human pancreas or islets, and in several
β-cell lines, however there are numbers of TRP channels expressed in pancreas whose
function are currently unknown (140)
3.1.1.1. Vanilloid receptor TRP channels (TRPV) in β-cells
Transient receptor potential vanilloid channels are cation channels and are
expressed in many tissues and cells, including neurons. As previously mentioned,
TRPV channels consist of six members. All subfamily members are heat-activated
proteins, however TRPV1 is also stimulated by low pH and has been identified as a
receptor for capsaicin, an active ingredient of chilli peppers. Endogenous compounds
have also been characterized as agonists for TRPV1 that includes fatty acid amides (N-
arachidonoyl serine and N-arachidonoyl taurine) (47). Akiba et al have shown that
activation of TRPV1 in β-cell by treatment with capsaicin or systematically injected
into rats caused insulin secretion (135). Activation of TRPVI channel might be
abolished by competitive action of capsazepine, a synthetic analogue that binds to
TRPV1 protein and inhibits capsaicin effect.
TRPV2 channel is also detected in insulinoma cell line MIN6, and mouse
pancreatic β-cell and intracellular Ca2+ increases were also observed upon TRPV2
activation (136, 141). TRPV2 channel is mainly localized in the cytoplasm, especially
32
to the endoplasmic reticulum (ER) and seems to be stimulated by level of insulin in
β-cell. In compare to TRPV1 and TRPV2, the TRPV4 channel is highly expressed in
epithelial cells and plays role in skin barrier (142). In addition to heat, TRPV4 as well
as TRPV1 is also activated by N-arachidonoyl taurine (47). TRPV channels subfamily
is a highly Ca2+ -permeable cation channels and activation of these receptors could
contribute to changes in intracellular Ca2+ concentrations ([Ca2+]i) and control of
membrane potential in many type of cells. It is known that continuous changes in
[Ca2+]i that occur in the form of oscillations are necessary for insulin release from
β-cells in the pancreas.
3.2. Calcium free ions signalling in the β-cells
The frequency of oscillations in calcium signals is essential to regulate insulin
secretion and so far at least three different types of Ca2+ oscillations in β-cells have
been described (143-145). Changes in the cytosolic free Ca2+ concentration generate
signals that regulate numerous cellular processes. Oscillations (pulsations) of free
calcium ions is preferable than continuously increase [Ca2+]i. Endlessness increase of
free calcium ions would surely damage cells from long exposure to very high
concentration of free calcium ions, whereas oscillates will lead to signalling pathways
activated by higher concentration of free calcium ions, and next intracellular Ca2+ will
return to homeostasis. In ‘resting’ β-cells, the [Ca2+]i is approximately 20-100 nM, and
outside the cells the Ca2+ concentration is 10 000 times higher ( approximately 1M).
Frequency of the oscillations is important for β-cells functions, high concentration of
an calcium channels agonist will lead to higher frequency of oscillations, and further,
either low concentration of agonist or high concentration of antagonists will lead to
low frequency of the pulsations. However, how cells ‘translate’ the increase of
intracellular Ca2+ in form of oscillations remains unknown.
Many particles in cell are involved in transduction of Ca2+ signalling. Cell
proteins are one of these units. Calmodulin is Ca2+ binding protein that regulates
intracellular concentration levels of free calcium ions, and further works as an
intracellular Ca2+ receptor (for review see (146), (147, 148)). Rapid increase of [Ca2+]i
is also buffered by membrane pumps and channels: voltage-gated Ca2+, and the
transient receptor potential (TRP) channels family, recently TRP vanilloid (TRPV)
33
channels have been shown to be involved in free calcium ion flux stimulated by
N-acyl taurines (Paper IV). Moreover other membrane-pumps are involved in induce
of frequency of the Ca2+ spikes. One of the most important pump is plasma membrane
Ca2+ ATPase (PMCA) that pumps out Ca2+ from the cytoplasm (149) and together with
other pumps/exchangers like Na+/Ca2+ plays a key role in a quick increase followed by
decrease of intracellular Ca2+ signalling in many cells, especially in β-cells of pancreas,
where free calcium ions signalling is essential for insulin secretion.
3.2. Insulin secretion
Pancreatic β-cells secrete insulin in response to various conditions in order to
control blood glucose levels. Elevation of blood glucose triggers the primary stimulus
for insulin secretion (for review see (150). In human, dominant form of insulin
secretions are pulses with intervals every about 5 minutes (151, 152), however β-cells
except of these intervals, against common opinion, secretes insulin constantly even
under starving conditions with significant increase after food intake. To enhance
insulin secretion, glucose from food uptake needs to be metabolized. In human,
glucose is transported into β-cells by glucose transporters GLUT1 and GLUT3 (153,
154). Glycolysis and metabolism of glucose increase an intracellular ATP/ADP ratio
(155, 156), which closes ATP-sensitive K+ channels (KATP) on the cell surface, which
initiate membrane depolarization (157). Closed KATP channels together with
depolarized membrane will lead to open voltage-dependent Ca2+ channels (VDCC).
The entry of Ca2+ through these channels increases the cytoplasmic free Ca2+
concentration. Triggers of free calcium ions flux will activate subcellular Ca2+ stores
like mitochondria and endoplasmic reticulum (ER). The Ca2+-induced Ca2+release
(CICR) processes in the ER in many impulsive cells, such as muscle cells, nerve cells,
and β-cells (158, 159). CICR requires activation of special receptors localized in the
ER and through which activation Ca2+ will be released to cytosol. The inositol
1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) are two main
families of Ca2+ channels in the ER, whose activation controls release Ca2+ to
cytoplasm. To avoid toxicity from long time exposure to high concentration of
intracellular free calcium ions plasma membrane Ca2+ ATPases (PMCA) pump out
Ca2+ from the cytoplasm and together with Na+/Ca2+ exchangers helps β-cells reach
34
‘resting’ stage. All described mechanisms head to increase of the cytoplasmic free Ca2+
concentration, leading to secretion of insulin-containing vesicles. (see Fig. 10).
Fig. 10. Various factors/molecules involved in Ca2+ signalling and direct to insulin secretion in
β-cell of Langerhans islets in pancreas. Glucose (Glu) with the blood stream is transporting to the
pancreas. Pancreatic β-cells express on their cell membrane small channel-molecules that transport
glucose into the β-cell called glucose transporters (GLUT). Glycolysis and metabolism of glucose in the
mitochondria increases cytosolic level ATP/ADP ration, which results in close of potassium
ATP-dependent K+ channels (K+ATP channels) and membrane depolarization, which lead to the opening
of voltage-gated Ca2+ channels. This cascade reaction increases calcium levels and together with
G-protein coupled receptors expressed in β-cells membrane, activates release of Ca2+ from intracellular
stores such as mitochondria and endoplasmic reticulum (via the Ca2+ - - induced Ca2+ release (CIRC)
pathway. The significant increase of free calcium ions in cytoplasm will lead to the exocytosis of insulin
granules (INS). After insulin has been released from the cells, plasma membrane Ca2+ ATPases (PMCA)
pump out Ca2+ from the cytoplasm and together with Na+/Ca2+ exchangers helps β-cells go back to the
‘rest cycle’.
35
Various hormonal factors, nutrients and drugs that signal through receptors or
channels can also influence the regulation of insulin secretion in pancreatic β-cells.
Several members of the TRP channels (139), including TRPM5 expressed within the
pancreatic islets of Langerhans, have been shown to mediate insulin secretion from
pancreatic β-cell lines (160). Other members of the TRP family of cation channels
known to function in regulation of insulin secretion are expressed in mouse, rat, or
human pancreas or islets, and in several β-cell lines, however there are a number of
TRP proteins whose functions are currently unknown. We studied the mechanisms of
insulin secretion trigger via TRP channels activation by N-acyl taurines (Paper IV)
and indentified pathway for insulin secretion enhance through TRPV1 channels (see
Fig. 11).
Fig. 11. N-acyl taurines mediate insulin secretion via the activation of TRPV1 channels
and/or other receptors in β-cell. N-acyl taurines (NAT) activate TRPV1 (and possibly other
36
TRP) channels in β-cell. N-arachidonoyl taurine (NATau) and N-oleoyl taurine (OLTau) via
TRPV1 and TRP-like Ca2+ channels enhance flux of free calcium ions into the cytoplasm of
β-cell. An increase of [Ca2+]i together with raised level of ATP/ADP ratio in the cytoplasm lead
to the closing of potassium ATP-dependent K+ channels (K+ATP channels) and membrane
depolarization, which result in the opening of voltage-gated Ca2+ channels. Flux of free calcium
ions into the cytoplasm will activate CICR mechanism for Ca2+ release form subcellular stores
like ER. Final step is the insulin granules exocytosis triggered by TRPV1 and TRP-like Ca2+
channels activation by N-acyl taurines.
37
II Aims of the thesis
This thesis aimed to identify enzymatic pathway(s) for production of N-acyl
glycines and how levels of these N-acyl glycines would be regulated by the post-
translational modification of the enzymes involved. Moreover, we were studying on
N-acyl taurines and their possible physiological functions in β-cells of Langerhans
islands of the pancreas. Taken together this work is dedicated to expanding field of
biosynthesis and physiological functions of N-acyl amino acids a novel signalling
lipids.
38
III Results and discussion
4. Characterization of human N-acyltransferase-like 2 (hGLYATL2)
PAPER I
The recent identification of N-acyl glycines in vivo has led to
discussions as to the metabolic pathways for their production.
In this study we have characterized the human glycine N-acyltransferase-like 2
(GLYATL2). The human GLYATL2 contains 294 amino acids and encodes protein of
~35 kDa. The protein contains a –KKYC motif at the carboxy terminal end and
–KKXX motifs have been suggested to be ER retention signals (161). Using green
fluorescent fusion protein (GFP) we show that GLYATL2 is localized to the
endoplasmic reticulum (ER) in HepG2 cells. Our data suggests that the presence of the
–KKYC at the C terminus of GLYATL2 is not directly involved in ER localization,
but that human GLYATL2 is likely associated to the ER. Using Real Time PCR, we
show that human GLYATL2 mRNA is mainly expressed in salivary gland, trachea,
brain, spinal cord and thyroid gland. Our results also show that recombinantly
expressed GLYATL2 can conjugate saturated and unsaturated medium- and long-chain
(C8-C20:4) acyl-CoA esters to glycine and the enzyme shows the highest glycine
conjugating activity with oleoyl-CoA to produce N-oleoyl glycine (Km – 4.4 µM, Vmax –
933.4 nmol/min/mg). We also show that human GLYATL2 is specific for glycine as
an acceptor molecule as it did not have any enzyme activity with taurine, serine or
alanine.
Our results identify a novel enzymatic pathway in human for production of
N-acyl glycines, mostly notably N-oleoyl glycine (see Fig. 7). GLYATL2 plays a
crucial role in production of medium- and long-chain, saturated and unsaturated N-acyl
glycines in human in tissues with barrier functions and also in brain and spinal cord,
which contribute significantly to a better understanding of the pathways, functions and
metabolism of N-acyl glycines as novel signalling molecules.
39
5. Human GLYATL2 is regulated by acetylation/deacetylation of
lysine 19
PAPER II
Lysine acetylation is emerging as a major PTM in non-nuclear proteins (89). A
murine mitochondrial homologue of the human GLYATL2 enzyme (identified in
Paper I), has been shown to be acetylated on position lysine 19 (K19) (107). As this
lysine residue is conserved also in human GLYATL2, we wanted to examine the
possible role of lysine 19 reversible acetylation on the enzyme activity of human
GLYATL2.
Mutation of lysine 19 in recombinant human GLYATL2 to arginine (R) and
glutamine (Q) shows that acetylation of K19 inhibits the enzyme and that deacetylation
is likely required for full activity. The K19R mutant (which retains a positive charge
and is a conserved substitution) resulted in an 80% reduction in enzyme activity. The
K19Q mutation (which abolishes the positive charge and therefore may mimic
acetylation) had a similar effect on enzyme activity, however enzyme functions of
hGLYATL2 were reduced by 50%.
Kinetic studies for N-oleoyl glycine production by the K19Q mutant show an
increase of Km and Vmax values (Km – 9.6 µM, Vmax – 1102.2 nmol/min/mg) comparing
to wild type (see PAPER I), whereas the K19R mutant showed a high increase in Km
and Vmax values (Km – 28.7 µM, Vmax – 2037.7 nmol/min/mg). The similar effects on
enzyme activity and kinetics parameters were observed for production of
N-arachidonoyl glycine by hGLYATL2, however no activity was detected for K19R
mutant.
Using tandem mass spectrometry (MS/MS) we were able to confirm that wild
type hGLYATL2 is non-acetylated protein on lysine 19 based on results from
recombinant expressed protein in both mammalian and bacterial systems. Furthermore,
treatment with NAM (deacetylase inhibitor) resulted in acetylation of Lysine 19.
In summary, our results show that acetylation/deacetylation of lysine 19 is
important for regulating the activity of hGLYATL2. Our study also links the PTM of
proteins with the production of N-acyl glycines thus allowing a rapid regulation of
human GLYATL2 in response to lipid signalling requirements.
40
6. Molecular characterization of novel human glycine
N-acyltransferases – hGLYATL1 and hGLYATL3
PAPER III
hGLYATL2 has been identified as an enzyme that conjugates medium- and
long-chain saturated and unsaturated acyl-CoA esters (C8:0 – C20:4) to glycine (Paper I)
and its enzymatic activity has been suggested to be regulated by post-translational
modification – a reversible acetylation on lysine on lysine 19 (Paper II). However,
several other N-acyl glycines have been identified in vivo but molecular pathways for
the biosynthesis of these have not been elucidated. An example of one bioactive
molecule is N-docosahexanoyl glycine (DOCGly), which has been identified in rats
(39), with the highest abundance in skin (~210 pmol/gram of dry tissue weight), then
in lung, spinal cord, kidney, liver and ovaries (~160 to ~50 pmol/gram of dry tissue
weight) (39). Recently Tan et al have identified 50 endogenous N-acyl amino acids
based on a targeted lipidomics approach (4). As was mentioned before, a gene cluster
of GLYATs exists in human and mouse and these gene products show 20 – 48%
sequence identity to each other in human, suggesting that they have different substrate
specificities and functions. We were therefore interested to work on the most
uncharacterized human glycine N-acyltransferases – hGLYATL1 and hGLYATL3.
Our results show that the GLYATL1 is localized to the ER, whereas
GLYATL3 localization remains unknown, however despite the fact of containing a
putative nuclear localization signal (NLS) is more likely not nuclear protein. The
RNAs data from commercially available human tissues are evidence for expression of
GLYATL1 mainly in liver and kidney, while GLYATL3 is expressed in pancreas and
liver and out of screened different cell lines it appeared to be expressed in human
monocytic cells (THP-1).
Using bioinformatics tools (I-TASSER, COFACTOR) we focused on
molecular characterization of these two proteins, showing predicted a
three-dimensional (3D) structures of hGLYATL1 and hGLYATL3, with included
prediction of binding site residues and we have discussed their potential role in
enzymes functions.
41
Taken together, molecular characterization of these two members of glycine
N-acyltransferase enzymes family is an essential and initiating step to further extensive
study on potential substrate candidates involved in the in vivo production of N-acyl
amino acids.
7. Other results on human glycine N-acyltransferase family
7.1. Three-dimensional (3D) structures prediction of hGLYAT proteins
Human GLYAT proteins have recently been extensively studied for better
understanding physiological processes they are taking part in, which is production of
N-acyl amino acids – signalling lipids with broth spectrum of physiological function,
like pain perception, regulation of body temperature also have important –anti-
inflammatory properties. What are the structures of human GLYAT proteins - enzymes
involved in production and regulation of N-acyl amino acids levels remain unknown.
Bioinformatics have identified predicted structures of these proteins, which is the first
step for better understanding mechanism behind production of signalling lipids by
these enzymes (unpublished results). Two bioinformatics programs have been use for
structure and function prediction I.TASSER and COFACTOR. I-TASSER server for
protein structure and function predictions, where 3D models are built based on
multiple-threading alignments by LOMETS and iterative TASSER assembly
simulations and then function insights are drove by matching the predicted models
with protein function databases (162, 163). The other program used was COFACTOR
a structure-based method for biological function annotation of protein molecules. We
provide COFACTOR with 3D-structural models of GLYAT proteins generated by use
of I-TASSER. Cofactor threaded the structure through three comprehensive function
libraries by local and global structure matches to identify functional sites and
homologies. Functional insights, including binding sites were derived from the best
functional homology template.
Using bioinformatics programs described above, we were able to predict
binding site residues, and where it was possible we generate predicted active site
residues of human GLYAT proteins (see Fig. 12 and Table 2).
42
43
Fig. 12. A three dimentional (3D) predicted structures of human GLYAT proteins. Panels A-H
shows predicted binding sites for hGLYATs. I-TASSER and COFACTOR programs used for this
analysis have established 16 predicted amino acids between 230 and 278 position that might act as a
binding residues in hGLYAT (A-B). 16 Amino acids have been predicted as binding site residues for
hGLYATL1 between 232 and 279 amino acid (C-D). 3D structure of hGLYATL2 shows only 4
predicted binding site residues between 228-238 positions (E-F) and hGLYATL3 exposes 16 amino
acid residues predicted as binding site position in protein sequence between 214 and 262 position.
Panels I-L shows predicted active sites for hGLYAT and hGLYATL2 proteins. Both hGLYAT (I-J) and
hGLYATL2 (K-L) show 2 amino acids in each sequence that may work as active residues, for hGLYAT
active site residues were predicted at position 244 and 271, whereas for hGLYATL2 at position 227 and
262.
44
Predicted binding site residues Predicted active
site residues
hGLYAT 230, 231, 232, 238, 239, 240, 241, 242,
243, 244, 263, 271, 272, 274, 275, 278
244, 271
hGLYATL1 232, 233, 234, 239, 240, 241, 242, 243,
244, 245, 264, 272, 273, 275, 276, 279
_
hGLYATL2 228, 233, 234, 237, 238 227, 262
hGLYATL3 214, 215, 216, 222, 223, 224, 225, 226,
227, 228, 247, 255, 256, 258, 259, 262
_
Table 2. Predicted binding site and active site residues in human GLYAT enzymes. Predictions
based on bioinformatics analysis by ITASSER and COFACTOR programs.
8. N-acyl taurines regulate insulin secretion in pancreatic β-cells
PAPER IV
Pancreatic β-cells release insulin as a response to increase of intracellular free
calcium ions. Frequency of oscillating Ca2+ is ‘translated’ by cell machinery as a signal
for insulin secretion. To date, many molecules including glucose have been described
as insulin secretagogues. This study has been focused on N-acyl taurines and their
function in insulin release mechanisms and whether or not action of N-arachidonoyl
taurine (NATau) and N-oleoyl taurine (OLTau) activate TRP channels, especially
TRPV1 channel.
Our results show that two members of N-acyl taurines: NATau and OLTau
increase the level of free calcium ions in the cytosol of the pancreatic β-cells. The
increase of free calcium ions has been observed in the form of oscillations, and high
frequency of pulsates will lead to wide insulin secretion. We have observed substantial
increase of insulin secretion from cells treated with NATau or OLTau. To exam the
possible molecular mechanism of insulin secretion from tested cell line (832/13 INS-
1), cells were treated with two inhibitors TRPV channels - capsazepine and ruthenium
red, as TRPV1 and TRPV4 have been suggested as receptor for N-arachidonoyl taurine
45
(45). Pre-incubation with TRPV1 channels antagonist - capsazepine followed by
treatment with NATau or OLTau results in significant (p<0.05) decrease of secreted
insulin to 50% and 62% respectively. Furthermore, pre-incubation with unspecific TRP
channels blocker ruthenium red followed by treatment with NATau or OLTau
significantly (p<0.05) reduce insulin exocytosis down to 55% and 72% respectively.
To summarize, our results have shown a novel pathway for insulin secretion
from pancreatic β-cells mediated by N-acyl taurines. N-arachidonoyl taurine as well as
N-oleoyl taurine administration can trigger a response for free calcium ions flux into
the cystosol of β-cells, finalized in enhanced insulin secretion. Presented data indicate
that the mechanism of insulin secretion increase leads through TRP, especially TRPV1
channel.
46
IV Future perspectives
Glycine conjugation has been the topic of renewed interest in recent years as a
result of the discovery of novel groups of compounds, the N-acyl amino acids. N-acyl
glycines are an example of N-acyl amino acids, and are considered as signalling
molecules, due to their physiological functions (activating G protein-coupled
receptors) in CNS and other peripheral tissues (23).
We are interested to analyze the level of N-acyl glycines in cerebrospinal fluid
(CSF) of patients with cognitive disorders and Alzheimer’s. This work will be carried
out using mass spectrometry methods and in theory may identify a new marker for
neurodegenerative diseases.
Furthermore, in recent years, there has been worldwide interest in developing
surface-active molecules that have both antimicrobial activity and are easily
biodegradable (164). The physiochemical and biological properties of N-acyl amino
acids have not been completely analyzed as potential antimicrobial and biodegradable
agents, although some published results suggest that some of these compounds may
have potential value as biostatic additives in commercial products (164, 165).
Investigation of the antimicrobial properties of N-acyl glycines might be considered as
another aspect of this study on enzymatic production of N-acyl glycines and their
functions in the human body. We are extensively working on GLYATL1 and
GLYATL3 and hope to identify the functions of these two proteins in N-acyl amino
acid production/metabolism.
Diabetes is a chronic disease that occurs when the pancreas does not produce
enough insulin, or when the body cannot effectively use the insulin it produces.
Hyperglycaemia, or raised blood sugar, is a common effect of uncontrolled diabetes
and over time leads to serious damage to many of the body's systems, especially the
nerves and blood vessels. Our resent study on N-acyl taurines as novel insulin
secretagogues in β-cells, raised a new questions about potential function of these
compounds in human islets and eventual propose to development of a new treatment
therapy against diabetes which affects approximately 350 million people worldwide
(World Health Organization).
47
V Popular science
48
VI Acknowledgements
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