Structural basis for the inhibition mechanism of HUman CSE and a study on c CBL complexes

1




Chapter I









Introduction







2

1.1 Gasotransmitters
Organ and cell function are orchestrated by a myriad of signal molecules that
belong to virtually all classes of substances including lipids, small and large peptides,
small organic and inorganic molecules (such as calcium, zinc and amino acids) and
numerous intermediates from metabolism. Interestingly, gaseous molecules also play
an important role as short-lived, local and often very potent signal molecules. These

molecules are called gaseous messengers or gasotransmitters which include nitric
oxide (NO), carbon monoxide (CO), and hydrogen sulfide (sulfide, H
2
S) (Figure 1).
While all three gases are important in a range of biological systems, NO and CO are
two major well-known and well studied gaseous signalling molecules in humans.

Figure 1. 3D ball representation of the structure of H
2
S, CO and NO respectively.

1.1.1 NO and CO
The role of nitric oxide signalling is well defined in processes such as neural
transmission and the dilation of blood vessels. NO is synthesized upon the cleavage of
L-arginine into L-citrulline by three distinct isoforms of NO synthase (NOS) within
the myocardium (Barouch et al., 2002). It has attracted much attention since its
3

discovery for its pleiotropic effects in myocardial function (Ziolo et al., 2008).
Appropriate levels of NO production are important in protecting an organ such as the
liver from ischemic damage. However sustained levels of NO production results in
direct tissue toxicity and contributes to the vascular collapse associated with septic
shock, whereas chronic expression of NO is associated with various carcinomas and
inflammatory conditions including juvenile diabetes, multiple sclerosis, arthritis and
ulcerative colitis (Hou et al., 1999).
The other important gasotransmitter, carbon monoxide (CO), is produced
physiologically by catabolism of heme to CO, iron, and biliverdin (Maines, 1997).
This reaction is catalyzed by heme oxygenase (HO) with reduction of NADPH. CO
stimulates guanylate cyclases but with much lower potency than NO (Wagner, 2009).
Abnormalities in its metabolism have been linked to a variety of diseases, including

neurodegenerations, hypertension, heart failure, and inflammation (Wu and Wang,
2005).






4

1.1.2 Hydrogen sulfide, H
2
S
Hydrogen sulfide (or hydrogen sulphide) is a colorless, flammable, water-soluble
gas with the characteristic smell of rotten eggs. For decades,
H
2
S
is known as a toxic
gas and as an environmental hazard. Only recently, H
2
S is found to be present in
mammalian tissues at various amounts. H
2
S can be produced from L‑cysteine, and
eventually converted to sulphite in the mitochondria by thiosulphate reductase, and
further oxidized to thiosulphate and sulphate by sulphite oxidase (Fiorucci et al., 2006;
Wang, 2002). These sulphates are then excreted in the urine (Kamoun, 2004). The
biological effects of H
2

S in mammalian cells are depicted below (Figure 2).


5


Figure 2. Some biological functions of H
2
S in mammalian cells.

Top left: known cellular targets of sulphide include cytochrome c oxidase and carbonic
anhydrase.

Top right: sulphide can participate in reactions yielding persulphide and polysulphide. Sulphide
can also bind to plasma proteins such as albumin, and it can activate ATP-activated potassium
(KATP) channels in the myocardium, vascular smooth muscle and cardiac myocytes. The
binding of sulphide to haemoglobin or myoglobin forms sulphaemoglobin or sulphmyoglobin.

Bottom panel: Some of the redox reactions that sulphide participates in can result in the
reduction of disulphide bonds, as well as reactions with various reactive oxygen and nitrogen
species, resulting in free-radical scavenging and antioxidant effects. Sulphide has also been
demonstrated to regulate cellular signal transduction pathways, resulting in alterations of the
expression of various genes and gene products including thioredoxin reductase and
interleukin-1β (IL1β). HO1: haem oxygenase 1 (adapted from Szabo, 2007).
6

1.2 Physiological roles of H
2
S
1.2.1 Smooth-muscle relaxation and neurotransmission

A major function of H
2
S in isolated organ systems is smooth-muscle relaxation.
Several groups have demonstrated that H
2
S relaxes rat aortic tissues in vitro (Fiorucci
et al., 2006; Hosoki et al., 1997; Zhao et al., 2001), possibly through the activation of
potassium channels (Figure 3). Intravenous bolus injection of H
2
S transiently
decreased rat blood pressure by 12-30 mmHg, but this effect was antagonized by prior
blockade of potassium channels (Zhao et al., 2001).

Figure 3. The relaxant effect of H
2
S on the aortic tissues. The aortic tisses are pre-contracted
with 20 or 100 mM KCl (adapted from Zhao et al, 2001).







7

At physiological concentrations, H
2
S selectively enhances NMDA
receptor-mediated responses and facilitates the induction of hippocampal long-term

potentiation (LTP) (Abe and Kimura, 1996) (Figure 4). The neuromodulator role of
H
2
S is believed to be important for the associative learning process of the brain.

Figure 4. Concentration dependency of the LTP-facilitating effect of Sodium Hydrosulfide
(NaHS). NaHS is an H
2
S releasing compound (adapted from Abe and Kimura, 1996).

1.2.2 Apoptosis, inflammation, cellular respiration inhibition and more
Multiple studies demonstrated the cytoprotective (antinecrotic or antiapoptotic)
effects of H
2
S at micromolar concentrations. Rinaldi et al found that H
2
S promoted
the survival of granulocytes. The pro-survival effect of H
2
S (Figure 5) was due to
inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation (Rinaldi et al.,
2006). In another study, H
2
S significantly inhibited peroxynitrite-mediated tyrosine
nitration and cytotoxicity (Whiteman et al., 2004). Anti-oxidative damage effect of
H
2
S was also observed in a study done by the same group (Whiteman et al., 2005). On
8

the other hand, it was found that high level of endogenous H
2
S concentrations caused
cellular apoptosis (Yang et al., 2004; Yang et al., 2006; Yang et al., 2007).

Figure 5. Effect of different inhibitors on the survival of purified human neutrophils. The
neutrophils are cultured for 24 h in the presence or absence of 1.83 mM NaHS. Control
samples were treated with DMSO alone (adapted from Rinaldi et al., 2006).
H
2
S suppresses the metabolic rate of the affected cell or organ at high
concentration (Lane, 2006). Cytochrome c oxidase activity is critical for cellular
respiration. H
2
S inhibits cellular respiration, at least in part by acting as an inhibitor of
cytochrome c oxidase (EC 1.9.3.1) via a reaction with its copper centre (Hill et al.,
1984). This inhibition has been implicated in induction of suspended animation in
house mouse, in which H
2
S inhalation induced marked decrease in metabolic rate,
followed by a loss of homeothermic control in which the animal's core body
temperature approached that of the environment (Blackstone et al., 2005) (Figure 6).
This state is readily reversible and does not appear to harm the animal, suggesting the
9

possibility of inducing suspended animation-like states for medical applications such
as ischemia and reperfusion injury, pyrexia, and other trauma.

Figure 6. Core Body Temperature and Metabolic Rate of mice exposed to H
2

S. (A) Relative
carbon dioxide production and oxygen consumption of mice exposed to 80 ppm of hydrogen
sulfide. (B) Core Body Temperature of mice during 6 hours of exposure to either 80 ppm of
hydrogen sulfide (black line) or the control atmosphere (gray line). The dotted line indicates
ambient temperature (adapted from Blackstone et al, 2005).
H
2
S at low concentration has anti-inflammatory effect, whereas at higher
concentration, it exerts pro-inflammatory effects (Li et al., 2005; Szabo, 2007;
Tripatara et al., 2008; Zanardo et al., 2006). In a study which used a total of 74 male
Wistar rats to investigate the effects of endogenous and exogenous hydrogen sulfide
in renal ischemia/reperfusion (Tripatara et al., 2008), administrating an irreversible
cystathionine gamma-lyase (CSE, also known as CGL) inhibitor,
D/L-propargylglycine (PAG), prevented the recovery of renal function after 45 min
ischemia and 72 h reperfusion. On the other hand, the hydrogen sulfide donor sodium
hydrosulfide (NaHS) attenuated renal dysfunction and injury caused by 45 min
ischemia and 6 h reperfusion (Figure 7). The protective effects were concluded to be
due to both anti-apoptotic and anti-inflammatory effects of hydrogen sulfide.
10


Figure 7. Exogenous H
2
S reduces the histological signs of injury caused by
ischemia/reperfusion injury (IRI) in rats. Shown is acute tubular necrosis score for control
(sham), renal IRI, or renal IRI with NaHS (100 mmol/kg, 2 ml/kg onto kidneys) administered 15
min before 45 min ischemia and 5 min before 6 h reperfusion (IRI NaHS). *P<0.05 vs IRI
(adapted from Tripatara et al., 2008).
Recently, H
2

S is also shown to facilitate erectile function in rabbits (Srilatha et al.,
2007) which suggests that it may, like NO, play a part in non-adrenergic,
non-cholinergic (NANC) transmission, presenting possible new therapeutic
opportunities for erectile dysfunction.

1.2.3 Diseases associated with reduced level of H
2
S activity
Due to the important physiological roles of H
2
S, physiological or in vivo
dysregulation of H
2
S level is shown to cause various diseases. Reduced H
2
S level is
observed at least in patients with Alzheimer's disease, coronary heart disease and in
spontaneously hypertensive rats. The brain hydrogen sulfide concentration is severely
decreased in Alzheimer's disease (AD) patients, leading to cognitive decline (Figure
11

8). (Eto et al., 2002). In coronary heart disease (CHD) patients, plasma sulfide levels
are also significantly lower and correlated with the severity of CHD (Jiang et al.,
2005). In another report, reduced production of endogenous H
2
S is found to be
important in the development of spontaneous hypertension in rats (Du et al., 2003).

Figure 8. Comparisons of endogenous H
2

S levels between AD and control brains. (A) H
2
S
level is decreased in AD brains. Brain H
2
S levels of each individual are shown. (B) Statistical
comparison of H
2
S levels between AD and control brains (adapted from Eto et al, 2002).

1.2.4 Diseases associated with increased level of H
2
S activity
Increased circulating sulfide concentrations are reported in Down syndrome, type
I diabetes mellitus and several circulatory shocks. (Kamoun, 2004; Kamoun et al.,
2003). In type I diabetes (Figure 9), beta cells of the pancreas produces an excessive
amount of H
2
S, leading to the death of beta cells and reduced production of insulin by
those that remain (Buschard et al., 2005; Wu et al., 2009).
12


Figure 9. Pancreatic islet production of H
2
S in 16-week rats. Propaygylglycine (PAG or PPG)
is an inhibitor of CSE. ZDF: Zucker diabetic fatty. *P<0.05 vs PPG-treated group; #P<0.05 vs
Zucker fatty (ZF) or Zucker lean (ZL) rats; n=3–5 for each group (adapted from Wu et al,
2009).
In a carrageenan model of paw oedema, an about 40% increase in

H
2
S-synthesizing activity is reported in paw homogenates (Bhatia et al., 2005a).
Pretreatment (i.p. 60 min before carrageenan) with DL-propargylglycine (PAG, 25-75
mg/kg), an inhibitor of the H
2
S synthesising enzyme cystathionine-gamma-lyase
(CSE), significantly reduces carrageenan-induced hindpaw oedema in a
dose-dependent manner (Figure 10). Similarly, in mouse model of pancreatitis and
lung injury (Bhatia et al., 2005b), haemorrhagic shock (Mok et al., 2004), cecal
ligation and puncture (Zhang et al., 2006), LPS induced endotoxic shock (Li et al.,
2005), and rat endotoximia (Collin et al., 2005), H
2
S levels in the plasma or relevant
organs is significantly increased to various degrees. In each case, pharmacological
inhibition of H
2
S biosynthesis is anti-inflammatory (Li et al., 2006).
13


Figure 10. PAG concentration effect in carrageenan induced hindpaw oedema. Results show
mean±s.e.m., n=10, *P<0.05, c.f. control, (administered intraplantar saline), +P<0.05, c.f.
carrageenan. Doses of PAG shown are mg/kg (i.p.) (adapted from Bhatia et al, 2005a).

1.2.5 Summary of H
2
S physiological importance
Previous sections highlight the importance of production of H
2

S in a number of
biological functions including vascular smooth muscle relaxation, neurotransmission,
cell proliferation and apoptosis, inflammation, and erectile function (Li et al., 2006; Li
and Moore, 2008; Szabo, 2007; Wagner, 2009). Dysregulation of H
2
S production is
14

implicated in many common diseases including Alzheimer's disease, coronary heart
disease, spontaneous hypertension, Down syndrome, diabetes, endotoxic, septic and
haemorrhagic shock. Therefore, it is of utmost importance to understand how H
2
S
production is regulated in the human body and how to enhance or reduce the level of
H
2
S in order to control these diseases.

15

1.3 In vivo hydrogen sulfide production
A wide range of organisms including bacteria and archaea (Pace, 1997),
non-mammalian vertebrates (Dombkowski et al., 2005) as well as mammals (Wang,
2003) have shown to produce and utilize H
2
S gas at physiological concentrations in
the range of 20-160µM (Abe and Kimura, 1996; Zhao et al., 2001). In human, two
pyridoxal-5’-phosphate (PLP)-dependent enzymes, cystathionine β-synthase (CBS EC
4.2.1.22) and cystathionine gamma-lyase (CSE EC 4.4.1.1) are largely responsible for
the in vivo production of H
2
S (Figure 11) at rates of 1–10 pmoles per second per mg
protein in homogenous tissue (Doeller et al., 2005). Since H
2
S is rapidly consumed
and degraded, there is very low extracellular concentration.

Figure 11. H
2
S production by CBS and CSE.
The substrate of CBS and CSE for H
2
S production, L‑cysteine, is derived from
many sources e.g. alimentary sources and endogenous proteins. It can even be
synthesized endogenously from L‑methionine through the trans-sulphuration pathway
16

(Fiorucci et al., 2006; Wang, 2002). CBS and CSE catalyze the hydrolysis of cysteine
via β-elimination to generate H
2
S. Other substrates for H
2
S production have been
reported elsewhere (Chen et al., 2004; Steegborn et al., 1999).
Human CBS is a Pyridoxal phosphate (PLP) (Figure 12) dependent enzyme
having a complex domain structure. PLP is a cofactor of this enzyme and is essential
for these reactions. CBS catalytic domain crystal structure has been solved and it
belongs to the β family of PLP dependent enzymes (Meier et al., 2001). CBS is the
predominant H
2
S-generating enzyme in the brain and nervous system and is also
highly expressed in liver and kidney (Miles and Kraus, 2004). The activity of CBS is
regulated presumably at the transcriptional level by glucocorticoids and cyclic AMP
and it can be inhibited by nitric oxide (NO) and carbon monoxide (CO) (Puranik et al.,
2006).

Figure 12. Chemical structure of PLP. PLP is a cofactor of CBS or CSE enzyme and is
essential for reactions.
The other H
2
S producing enzyme, CSE, is a PLP dependent enzyme mainly
responsible for the production of H
2
S outside of the nervous system especially in liver
and in vascular and non-vascular smooth muscle tissues (Szabo, 2007). Low levels of
CSE are also detectable in the small intestine and stomach of rodents (Fiorucci et al.,
17

2006). Its regulatory mechanisms are not well understood. Several studies have shown
that inhibition of CSE activity, especially in the smooth muscle tissue, has promising
therapeutic applications (Collin et al., 2005; Li et al., 2005; Mok et al., 2004; Yusuf et
al., 2005; Zhang et al., 2006).

















18

1.4 CSE: the main vascular H
2
S producing enzyme
1.4.1 Primary sequence and structural information
CSE gene is localized on human chromosome 1. This gene is well conserved
from yeast to mammals. Notebly, there is no CSE protein identified in becteria and

plants (Steegborn et al, 1999). The human CSE (hCSE) sequence has significant
amino acid identity to the rat (85%) and yeast (50%) enzymes. Despite there is no
seuqnce homology between human CBS and CSE both are involved in H
2
S
production. Human CSE consists of 405 amino acids, has a molecular weight of about
45 kD, an isoelectric point around 6.2, and exists as a cytosolic, functional tetramer.
CSE belongs to the trans-sulfuration enzymes gamma family, along with
cystathionine gamma synthase and cystathionine beta lyase. Structure homolog of
CSE from Saccharomyces cerevisiae reveals insights into the enzymatic specificity
among the different family members (Messerschmidt et al in 2003). However，the
structure of human CSE was not available until we solved it.

1.4.2 Reactions catalyzed by CSE
Besides catalysing the elimination reactions from L-cysteine, to produce pyruvate,
NH3 and H
2
S, Cystathionine gamma-lyase also breaks down cystathionine into
cysteine and α-ketobutyrate (Figure 13). Steegborn et al in 1999 studied the kinetics
of hCSE and reported the activity of this enzyme towards L-cystathionine, L-cystine
19

and L-cysteine, and performed several inhibition studies using CSE inhibitors
including PAG.

Figure 13. Reactions catalyzed by CSE. CSE converts L-cystathionine into L-cysteine,
α-ketobutyrate and ammonia in the reverse transsulfuration pathway via an α,γ-elimination
reaction. This enzyme can also utilize L-cysteine as a substrate in an α,β-elimination reaction
to produce H
2

S, pyruvate and ammonia (adapted from Huang et al., 2010).

1.4.3 CSE regulation in vivo
CSE is phosphorylated upon DNA damage, probably mediated by ATM (ataxia
telangiectasia, mutated) and ATR (ATM and Rad3-related) (Matsuoka et al., 2007).
Interaction with Calmodulin in a calcium-dependent manner has been reported,
resulting in an upregulation of CSE activity (Yang et al., 2008). Recently, Renga et al
(2009) reported that liver expression of CSE is regulated by bile acids by means of a
farnesoid X receptor (FXR) mediated mechanism. Western blotting, qualitative and
quantitative polymerase chain reaction, as well as immunohistochemical analysis,
showed that the expression of CSE in HepG2 cells and in mice was induced by
treatment with a farnesoid X receptor ligand (Figure 14). Administration of
20

6-ethyl-chenodeoxycholic acid (6E-CDCA), a synthetic FXR ligand, to rats protected
increased CSE expression level, increased H
2
S generation, reduced portal pressure
and attenuated the endothelial dysfunction of isolated and perfused cirrhotic rat livers.
These results provide a new molecular explanation for the pathophysiology of portal
hypertension (Renga et al., 2009).

Figure 14. CSE expression/activity is regulated with an FXR ligand in vivo. FXR +/+ and FXR
-/- mice were treated for 3 days with vehicle or with 6E-CDCA 5 mg/kg body weight.

A: Total RNA from liver of FXR +/+ and FXR -/- mice was subjected to real-time PCR
quantification of CSE gene expression. a P < 0.05 versus FXR +/+ control mice;

B: Livers from FXR +/+ and FXR -/- mice were homogenized in cold PBS to evaluate CSE
activity. a P < 0.05 versus FXR +/+ control mice. c P < 0.05 versus FXR -/- control mice;

C: Livers from FXR +/+ and FXR -/- mice were homogenized in cold PBS to evaluate H
2
S
production. a P < 0.05 versus FXR +/+ control mice (adapted from Renga et al., 2009).

1.4.4 Natural CSE mutations
Natural, non-active CSE mutations are associated with cystathioninuria, a disease
characterized by accumulation of cystathionine in blood, tissue and urine, and
21

sometimes associated with mental retardation (Tang et al., 2006; Wang and Hegele,
2003). The PLP content in the two natural CSE mutants, T67I and Q240E, were about
4-fold and 80-fold lower than that of wild-type enzyme, respectively. Pre-incubation
of the T67I mutant with PLP restored activity to wild-type levels while the same
treatment resulted in only partial restoration of activity of the Q240E mutant (Table
1). These results reveal that both mutations weaken the affinity for PLP and suggest
that cystathionuric patients with these mutations should be responsive to pyridoxine
therapy. (Zhu et al., 2008).
Table 1. Comparison of the kinetic parameters for the polymorphic variants of CSE (adapted
from Zhu et al, 2008).



22

1.5 Recent studies on CSE down regulation
1.5.1 CSE Knockout mice
Hydrogen sulfide (H
2

S) was demonstrated to be physiologically generated by
CSE and genetic deletion of this enzyme in mice markedly reduced H
2
S levels in the
serum, heart, aorta, and other tissues (Yang et al., 2008). Mutant mice lacking CSE
displayed pronounced hypertension (Figure 15) with diminished
endothelium-dependent vasorelaxation. Taken together, their finding provided direct
evidence that CSE is an important regulator of vasodilation and blood pressure.

Figure 15. Higher blood pressure in CSE male knockout mice. A) Reduced serum H
2
S level in
CSE–/– mice and CSE–/+ mice (n = 8 to 10). B) Age-dependent increase in blood pressure of
CSE–/– mice and CSE–/+ mice (n = 12) (adapted from Yang, Wu et al. 2008).

1.5.2 Studies using CSE inhibitors: BCA and PAG
Studies of several of animal disease models have revealed that pre- or
post-treatment with inhibitors of CSE, such as DL-propargylglycine (PAG) or
23

β-cyanoalanine (BCA) (Figure 16) not only inhibits tissue H
2
S production but also
reduces the severity of the disease. In a typical study, Mok et al showed that
pre-treatment (30 min before shock) or post-treatment (60 min after shock) with BCA
or PAG, abolished the rise in plasma H
2
S in animals exposed to 60 min haemorrhagic
shock and prevented the augmented biosynthesis of H
2

S from cysteine in liver.
Similarly, pre-treatment of animals with PAG or BCA produced a rapid, partial
restoration in mean arterial blood pressure (MAP) and heart rate (HR) (Mok et al.,
2004). In two other studies, PAG and BCA have been shown to reduce inflammation
level in rodent models (Bhatia et al., 2005a; Bhatia et al., 2005b).

Figure 16. Chemical structure of BCA and PAG. BCA (β-cyanoalanine) (left) and PAG
(dl-propylargylglycine) (right) are commonly used as inhibitors of H
2
S biosynthesis, although
they have low potency and selectivity, and limited cell-membrane permeability. This figure is
generated by Chemdraw.
In several other studies, inhibition of CSE by either BCA or PAG increased the
disease severity, explained by the cytoprotective effect of H
2
S. Particularly,
administration of PAG (50 mg/kg) significantly increased infarct (local tissue death)
size caused by 15 min of myocardial ischemia (Sivarajah et al., 2006). The delayed
cardioprotection afforded by endotoxin was abolished by PAG. These findings
24

highlight the importance of CSE-mediated production of H
2
S in regulating a number
of human body functions. For more complete overview of the related studies, please
refer to the table below (Table 2).
Table 2. Effect of inhibiting CSE in animal models of disease(adapted from Szabo, 2007)

25

1.6 Therapeutic potential of human CSE
Hydrogen sulfide (H
2
S), produced by CSE, is a novel regulator of important
physiological functions such as neurotransmission, arterial diameter, blood flow and
leukocyte adhesion (Wagner, 2009). Emerging data on the biological effects of H
2
S
support a basic approach for the development of inhibitors/activators of CSE.
Currently no small molecule activators of CSE are available. BCA and PAG are
probably the best available inhibitors for CSE. These compounds, although of low
potency, low selectivity and limited cell-membrane permeability (Burnett et al., 1980;
Marcotte and Walsh, 1976), are widely used in animal studies. However, for
therapeutic application, there is a need to design more specific and potent
inhibitors/activators for CSE enzyme.
Towards understanding the mechanism of H
2
S biosynthesis and designing new
inhibitor/activator, we have determined the crystal structures of human
cystathionine-γ-lyase in the apo form (apo-CSE), complexed with PLP (CSE-PLP)
and with its inhibitor PAG. From the CSE crystal structures, biochemical and
biophysical studies, molecular details of CSE mediated production of H
2
S and
inhibition of H
2
S production by PAG is discussed. Please refer to chapter II of this
thesis for the details of this project.
Moreover, H
2

S itself is a signal molecule and plays a significant role in regulating
signal transduction. The following part of this chapter gives a brief introduction of
signal transduction and several proteins involved in signalling, particularly c-Cbl, a