From the Department of Genetics, Microbiology and Toxicology Stockholm University
Biosynthesis and physiological functions of N-acyl amino acids
Dominik Paweł Waluk
Stockholm 2012
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
3 ABSTRACT
4 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)
5 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
6 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
7 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.
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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
9 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
10 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).
11 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.
A
O
OH N H
O N-octanoyl glycine (N-capryloyl glycine)
B O
OH N H
N-decanoyl glycine O (N-caproyl glycine)
C O
OH N H
N-dodecanoyl glycine O (N-lauroyl glycine)
D O
OH N H
N-tetradecanoyl glycine O (N-myristoyl glycine)
12 E O
OH N H
O
N-hexadecanoyl glycine (N-palmitoyl glycine)
O F OH N H
O
N-9-hexadecenoyl glycine (N-palmitoleoyl glycine)
G O
OH N H
O
N-octadecanoyl glycine (N-stearoyl glycine) H O
OH N H
O
N-9-octadecenoyl glycine (N-oleoyl glycine)
I O
OH N H
O
N-9,12-octadecadienoyl glycine (N-linoleoyl glycine)
13 J O
OH N H
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
14 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
O O A OH S NH O
N-9-octadecenoyl taurine (N-oleoyl taurine)
B O O OH S N H O
N-9,12-octadecadienoyl taurine (N-linoleoyl taurine)
15
C O O
OH S N H O
N-5,8,11,14-eicosatetraenoyl taurine (N-arachidonoyl taurine)
D O OH
S O
N
O
N-4,7,10,13,16,19-docosahexaenoyl taurine (N-cervonoyl taurine)
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
16 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.
17 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).
18 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).
19 O
OH
FREE ARACHIDONIC ACID OH
H2N ALCOHOL DEHYDROGENASE ETHANOLAMINE (ADH7) O OH FAAH N H
ANANDAMIDE
O H H2N N OH H O ETHANOLAMINE O ARACHIDONOYL GLYCINAL OH N H O
ARACHIDONOYL GLYCINE O
GLYATL2 H2N OH ALDEHYDE DEHYDROGENASE GLYCINE
O FAAH CoA O
H2N ARACHIDONOYL-CoA OH GLYCINE
H2O2 PEPTIDYLGLYCINE O α-AMIDATING
MONOOXYGENASE H2N cytochrome c OH GLYCINE O OH OH N H O
O ARACHIDONOYL-α-HYDROXYGLYCINE O H OH
NH2 O GLYOXYLIC ACID ARACHIDONAMIDE
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.
20 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.
21 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).
5Exons 5Exons 5Exons 5Exons
GLYAT GLYATL2 GLYATL1 GLYATL3
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
22 O
S CoA OH + H2N
O
Oleoyl-CoA Glycine
GLYATL2 glycine N- acyltransferase like 2 E.C.2.3.1.13
O
OH N H + HS CoA O
N-Oleoyl glycine
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
23 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
24 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.
H O
H3N N COO
O
CH3 H3C O H2N O SCoA
HDACs HATs
H O
OH H3N N COO CoAS
HN CH3
O
25 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.
26 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
27 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)).
28
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
29 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
30 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
31 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 2+ 2+ contribute to changes in intracellular Ca concentrations ([Ca ]i) and control of membrane potential in many type of cells. It is known that continuous changes in 2+ [Ca ]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 2+ calcium ions is preferable than continuously increase [Ca ]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 2+ return to homeostasis. In ‘resting’ β-cells, the [Ca ]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 2+ 2+ intracellular Ca receptor (for review see (146), (147, 148)). Rapid increase of [Ca ]i is also buffered by membrane pumps and channels: voltage-gated Ca2+, and the transient receptor potential (TRP) channels family, recently TRP vanilloid (TRPV)
32 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
33 ‘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’.
34 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
35 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 2+ β-cell. An increase of [Ca ]i together with raised level of ATP/ADP ratio in the cytoplasm lead + + to the closing of potassium ATP-dependent K channels (KATP 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.
36 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.
37 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.
38 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.
39
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.
40 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).
41
42
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.
43 Predicted binding site residues Predicted active site residues hGLYAT 230, 231, 232, 238, 239, 240, 241, 242, 244, 271 243, 244, 263, 271, 272, 274, 275, 278 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
44 (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.
45 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).
46 V Popular science
47 VI Acknowledgements VII References
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