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

1.7.4 Receptor tyrosine kinase EGFR EGFR epidermal growth factor receptor is a transmembrane RTK which can be activated by binding of its specific ligands, including epidermal growth fa

Chapter I

Introduction

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, H2S) (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 H2 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

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)

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, H2S is found to be present in

mammalian tissues at various amounts H2S 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 H2S in mammalian cells are depicted below (Figure 2)

Figure 2 Some biological functions of H2S 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-1 β (IL1 β ) HO1: haem oxygenase 1 (adapted from Szabo, 2007)

1.2 Physiological roles of H2S

1.2.1 Smooth-muscle relaxation and neurotransmission

A major function of H2S in isolated organ systems is smooth-muscle relaxation Several groups have demonstrated that H2S 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 H2S 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 H2 S on the aortic tissues The aortic tisses are pre-contracted with 20 or 100 mM KCl (adapted from Zhao et al, 2001)

At physiological concentrations, H2S 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

H2S 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

1.2.2 Apoptosis, inflammation, cellular respiration inhibition and more

Multiple studies demonstrated the cytoprotective (antinecrotic or antiapoptotic)

effects of H2S at micromolar concentrations Rinaldi et al found that H2S promoted

the survival of granulocytes The pro-survival effect of H2S (Figure 5) was due to

inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation (Rinaldi et al.,

2006) In another study, H2S significantly inhibited peroxynitrite-mediated tyrosine

nitration and cytotoxicity (Whiteman et al., 2004) Anti-oxidative damage effect of

H2S was also observed in a study done by the same group (Whiteman et al., 2005) On

the other hand, it was found that high level of endogenous H2S 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)

H2S suppresses the metabolic rate of the affected cell or organ at high concentration (Lane, 2006) Cytochrome c oxidase activity is critical for cellular

respiration H2S 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 H2S 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

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 H2 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)

H2S 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

Figure 7 Exogenous H2 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, H2S 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 H2S, physiological or in vivo

dysregulation of H2S level is shown to cause various diseases Reduced H2S 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

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 H2S is found to be

important in the development of spontaneous hypertension in rats (Du et al., 2003)

Figure 8 Comparisons of endogenous H2 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 2S 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 H2S, leading to the death of beta cells and reduced production of insulin by

those that remain (Buschard et al., 2005; Wu et al., 2009)

Figure 9 Pancreatic islet production of H2 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

H2S-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 H2S 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), H2S levels in the plasma or relevant

organs is significantly increased to various degrees In each case, pharmacological

inhibition of H2S biosynthesis is anti-inflammatory (Li et al., 2006)

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 H2S 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 H2S production is

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 H2S

production is regulated in the human body and how to enhance or reduce the level of

H2S in order to control these diseases

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 H2S 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 H2S (Figure 11) at rates of 1–10 pmoles per second per mg

protein in homogenous tissue (Doeller et al., 2005) Since H2S is rapidly consumed and degraded, there is very low extracellular concentration

Figure 11 H2 S production by CBS and CSE

The substrate of CBS and CSE for H2S 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

(Fiorucci et al., 2006; Wang, 2002) CBS and CSE catalyze the hydrolysis of cysteine

via β-elimination to generate H2S Other substrates for H2S 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 H2S-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 H2S producing enzyme, CSE, is a PLP dependent enzyme mainly

responsible for the production of H2S outside of the nervous system especially in liver and in vascular and non-vascular smooth muscle tissues (Szabo, 2007) Low levels of

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)

1.4 CSE: the main vascular H2S 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 H2S

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 H2S, 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

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

6-ethyl-chenodeoxycholic acid (6E-CDCA), a synthetic FXR ligand, to rats protected

increased CSE expression level, increased H2S 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

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)

1.5 Recent studies on CSE down regulation

1.5.1 CSE Knockout mice

Hydrogen sulfide (H2S) was demonstrated to be physiologically generated by

CSE and genetic deletion of this enzyme in mice markedly reduced H2S 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 H2 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

β-cyanoalanine (BCA) (Figure 16) not only inhibits tissue H2S 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 H2S in animals exposed to 60 min haemorrhagic

shock and prevented the augmented biosynthesis of H2S 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 H2S 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

highlight the importance of CSE-mediated production of H2S 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)

1.6 Therapeutic potential of human CSE

Hydrogen sulfide (H2S), 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 H2S 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 H2S 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 H2S and

inhibition of H2S production by PAG is discussed Please refer to chapter II of this

thesis for the details of this project

Moreover, H2S 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

protein involved in ubiquitination and recognition of the phosphotyrosine containing

motifs

1.7 Signal transduction

1.7.1 Signalling and tyrosine phosphorylation

Signaling or signal transduction is a mechanism that converts a

mechanical/chemical stimulus to a cell into a specific cellular response Numerous

molecules, linked in a series of intricate intracellular networks, are employed to

conduct signal transduction Particularly, protein phosphorylation is one of the

essential ways that play a wide role in cellular processes

Phosphorylation is the addition of a phosphate (PO4) group to a protein or other

organic molecule Protein phosphorylation can occur via serine (Ser), threonine (Thr)

and tyrosine (Tyr) residues Protein kinases are the enzymes that transfer the

phosphate of ATP to the hydroxyl group of a protein substrate, and phosphatases

reverse the process While phosphorylation in general is common, tyrosine

phosphorylation is rare and more critical in regulating cell responses ranging from cell

growth to death

1.7.2 Receptor tyrosine kinases and the MAPK pathway

Receptor tyrosine kinases (RTKs) are the high-affinity cell surface receptors for

many polypeptide growth factors, cytokines, and hormones Of the ninety unique

tyrosine kinase genes known in the human genome, 58 encode receptor tyrosine

kinase proteins (Robinson et al., 2000) Receptor tyrosine kinases have been shown

not only to be key regulators of normal cellular processes but also to play a key role in

the development and progression of many types of cancer (Zwick et al., 2001) When

a growth factor binds to the extracellular domain of an RTK, its dimerization is

triggered with other adjacent RTKs and the receptor is activated through

phosphorylating tyrosine residues on themselves, a process called

autophosphorylation RTK activation leads to downstream signal transduction

pathways, such as the well conserved mitogen activated protein kinase (MAPK)

signalling cascade (Zwick et al., 2001)

Mitogen Activated Protein Kinase (MAPK) pathways make up the backbone of

signal transduction machinery within the cells It can be activated by mitogens,

cytokines or even physical and chemical stressors such as UV irradiation, heat and

osmotic shock In general, MAPK activation involves the sequential phosphorylation

and activation of three kinases, namely, MAP kinase kinase kinase (MAPKKK or

MAP3K or MEKK), MAP kinase kinase (MAPKK or MAP2K or MEK) and MAP

kinase (MAPK) (Figure 17) Phosphorylated/activated MAPKs can migrate to the

nucleus to recognize transcription factors (Lewis et al., 1998) and control almost all

cellular processes ranging from gene expression to cell death (Chiang et al., 2001) In

mammals, MAPK is further divided into at least 4 distinctly regulated groups,

ERK1/2 (classical MAPK), JNK, p38, ERK5, depending on which MAPK is activated

In particular, activation of p38 MAPK often results in cell apoptosis or differentiation

Figure 17 Simplified MAPK signalling pathway Three kinases are sequentially

activated through phosphorylation An example of classical MAPK pathway is given

on the right side

1.7.4 Receptor tyrosine kinase EGFR

EGFR (epidermal growth factor receptor) is a transmembrane RTK which can be

activated by binding of its specific ligands, including epidermal growth factor and

transforming growth factor α (TGFα) Upon activation, EGFR dimerization occurs

and stimulates its intrinsic intracellular protein-tyrosine kinase activity As a result,

autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of

EGFR occurs These include Y992, Y1045, Y1068, Y1148 and Y1173 (Downward et

al., 1984) Activated EGFR then recruits several other proteins that associate with the

phosphorylated tyrosines through their phosphotyrosine-binding domains, to initiate

several signal transduction cascades, principally the ERK1/2, Akt and JNK pathways,

leading to DNA synthesis and cell proliferation (Oda et al., 2005)

Cancer arises when the activity of signalling proteins such as the EGFR are

breast cancer and lung cancer There is a strong correlation between high levels of

EGFR in breast tumors and the aggressive potential of the tumor Increased EGFR is

implicated in metastasis, poor efficiency of tamoxifen (EGFR antibody) therapy, and

low survival rate (Alvarez et al., 2010; Yoshida et al 2010)

1.7.5 RTK regulation through Spry2

The Sprouty family of proteins was first discovered as a negative feedback

regulator of fibroblast growth factor receptor (FGFR) (Hacohen et al., 1998) Since

then, four human Sprouty (Spry) isoforms were discovered They were able to form

heterodimers (with its isoforms) and homodimers for enhanced inhibition properties

(Bundschu et al., 2006), and further finding reveal that it also regulate the Ras/ERK

signal from receptors such as epidermal growth factor (EGF), (Frank et al., 2009) and

hepatocyte growth factor (HGF) (Lee et al., 2004) Ironically, Spry2 can also enhance

EGFR signalling through c-Cbl, a protein which can ubiquitinate EGFR for

endocytosis (Haglund et al., 2005) Sprouty2 gene expression is silenced or repressed

in cancers of the breast, liver, lung, prostate (Frank et al., 2009), and lymphoma

(Sanchez et al., 2008)

1.7.6 Negative RTK regulation through ubiquitination and receptor endocytosis

Ubiquitination is a negative feedback mechanism of RTK signalling For example,

activated EGFR is rapidly cleared from the surface by covalently attaching of

ubiquitin to the receptor Polyubiquitination generally results in proteosomal

degradation of protein (Ciechanover, 2005) Monoubiquitination of membrane bound

receptor tyrosine kinase, on the other hand, usually results in endocytosis of the

receptors (e.g EGFR, CSF1R and insulin receptors), followed by either lysosomal

degradation or receptor recycling (Hicke, 2001) Besides this function, ubiquitination

also controls the stability, function and intracellular localization of a wide variety of

proteins

The process of marking a protein with ubiquitin (ubiquitylation or ubiquitination)

consists of a series of steps (Figure 18) Ubiquitin is a small regulatory protein that

has been found in almost all tissues (ubiquitously) of eukaryotic organisms First,

ubiquitin is activated in a two-step reaction by an E1 ubiquitin-activating enzyme in a

process requiring ATP as an energy source Next, the ubiquitin is transferred from E1

to the active site cysteine of an ubiquitin-conjugating enzyme E2 via a trans

(thio)esterification reaction The final step of the ubiquitination cascade creates an

isopeptide bond between a lysine of the target protein and the C-terminal glycine of

ubiquitin In general, this step requires the activity of one of the hundreds of E3

ubiquitin-protein ligases (often termed simply ubiquitin ligase) E3 enzymes function

as the substrate recognition modules of the system and are capable of interaction with

both E2 and substrate

Figure 18 Schematic diagram of the ubiquitination system E3 enzymes function as the

substrate recognition modules of the system and are capable of interaction with both E2 and substrate S stands for substrate Dotted lines represent protein-protein interactions

1.7.7 E3 ubiquitin ligase c-Cbl

c-Cbl is an E3 ubiquitin ligase and multi-functional adapter protein (Tsygankov et

al., 2001) This protein plays an important role in controlling cell proliferation,

differentiation, and cell morphology as well as pathologies in response to different

stimuli, for example, growth factors, hormones and cytokines (Dikic et al., 2003) In

addition, pathological changes in c-Cbl have been shown to be involved in the

development of human diseases such as immune disease, diabetes and cancer

(Bachmaier et al., 2000)

There are three mammalian members of Cbl proteins, c-Cbl, Cbl-b and Cbl-c

c-Cbl (often referred to as Cbl) is the ubiquitous member in mammals and is the most

well studied target Cbl-b and Cbl-c are only present in certain tissues (such as

endodermally derived tissue) and have lower expression level (Keane et al., 1999)

Mutant c-Cbl has cell transforming properties The first discovered mouse Cbl mutant,

v-Cbl, had lost its ubiquitination ability and is responsible for the disease Caspas

B-cell lineage Lymphomas, hence its name Cbl (Langdon et al., 1989)

Besides mammals, Cbl has been found in a variety of organisms, e.g Drosophila

and C elegans (Hime et al., 1997; Yoon et al., 1995) Sequence homology study of

these sequences has found a particularly conserved N-terminal region (Figure 19)

The conserved region consists of two domains, Tyrosine Kinase Binding (TKB)

domain and Ring Finger (RF) domain, separated by a linker sequence Crystal

structures of c-Cbl TKB domain with ZAP 70 and APS peptides (Hu and Hubbard,

2005) revealed that it comprises of three sub-domains within the TKB domain, a

divergent SH2 domain which binds to phosphorylated tyrosine, a four-helix bundle

(4H) which packs against SH2 domain and completes the phosphotyrosine binding

pocket, and a calcium binding EF hand which wedges between the SH2 and 4H

domain The RING domain recruits ubiquitin–conjugating enzyme (E2), and catalyzes

the transfers of ubiquitin to the target protein (Zheng et al., 2000) The C-terminal of

c-Cbl contains a proline rich region, several serine and tyrosine phosphorylation sites,

a leucine zipper/ubiquitin associated domain (LZ/UBA) The presence of proline rich

regions and many serine and tyrosine phosphorylation sites enables c-Cbl to interact

with hundreds of different proteins especially the tyrosine kinase signalling proteins

and to regulate those signalling pathways (Schmidt and Dikic, 2005)

LZ/UBA

S S S Y Y Y Proline rich

Ring Finger SH2

EF

4H

Figure 19 Domain architecture of the full length Cbl The Cbl protein is a multi-domain protein

The N-terminal domain is conserved The C-terminal domain contains several serine and tyrosine phosphorylation sites (SSS and YYY), which greatly increases the functional complexity of this protein

1.7.8 c-Cbl, EGFR and Spry2 interactions

One of the most well studied functions of c-Cbl is the down regulation of

epithelial growth factor receptor (EGFR) through receptor endocytosis (Galisteo et al.,

1995; Tanaka et al., 1995) The TKB binding site in EGFR has been mapped to

Tyr1069 (Waterman et al., 2002) The oxidative stress activated EGFR, which fails to

be phosphorylated in Tyr1069, does not undergo c-Cbl-mediated ubiquitination and

endocytosis (Ravid et al., 2002) c-Cbl mediates EGFR endocytosis by functioning as

an adapter protein and as an ubiquitin ligase (Dikic et al., 2003) As an adaptor, c-Cbl

C-terminal sequence recruits CIN85/endophilins and CD2AP which work together

representing dynamin-dependent, clathrin-mediated receptor (EGFR) endocytosis

(Lynch et al., 2003; Soubeyran et al., 2002) At the same time, c-Cbl as an E3

ubiquitin ligase mono-ubiquitinates EGFR, which recruits ESP15 or Epsin and

undergoes lipid raft-dependent receptor endocytosis (Sigismund et al., 2005) These

two routes of endocytosis are likely to be synergetic to ensure the down-regulation of

EGFR (Dikic et al., 2003)

It is demonstrated that Spry2 is tyrosine-phosphorylated upon stimulation by

either FGF or EGF and subsequently binds endogenous c-Cbl with high affinity (Fong

et al., 2003) A conserved motif on Spry2, together with phosphorylation on tyrosine

55, is required for its enhanced interaction with the TKB domain of c-Cbl Spry2

mutant (Y55F) that did not bind with c-Cbl failed to retain EGF receptors on the cell

surface (Fong et al., 2003) Although the functional implication of Spry2 interaction

with Cbl is still poorly understood, it may serve as a mechanism for the

downregulation of Spry2 during receptor tyrosine kinase signaling (Hall et al., 2003)

1.7.9 Additional phosphorylation in the c-Cbl recruiting motif (N/D)XXpY

(S/T)XXP

The crystal structures of c-Cbl TKB domain with c-Cbl binding phosphorylated

peptides from Spry2, EGFR, Met, Syk were previously solved (Ng et al., 2008) The

conserved TKB domain of c-Cbl binds to phosphotyrosine with three conserved

sequence motifs: (D/N)XpY (S/T)XXP or RpY (S/T)XXP or DpYR An intrapeptidyl

bond between phosphotyrosine and an adjacent D/N or R was shown to be

indispensable for the binding In several other studies, it is suggested that not only can

the tyrosines be phosphorylated, but the adjacent partially conserved serine or

threonine in the c-Cbl-recruiting site of EGFR or Spry2 could also be phosphorylated

(Heisermann and Gill, 1988; Sweet et al., 2008)

Phosphorylation of serine residues (1070 and 1071) in the conserved motif RYp

(S/T)XXP of EGFR was reported as early as 1988 (Heisermann and Gill, 1988)

Phosphorylation of these residues is shown to be involved in receptor desensitization

(Countaway et al., 1992), internalization (Gamou and Shimizu, 1994), and kinase

activity inhibition (Feinmesser et al., 1999) It is shown that this pathway is

independent of tyrosine kinase activity Recently, phosphorylation of Spry2

Threonine 56, which is adjacent to pY55, was reported (Sweet et al., 2008) However,

the function of this additional phosphorylation is not well understood Since these

additional phosphorylation sites in EGFR and Spry2 located close to the essential

phosphotyrosine in the c-Cbl recognition motif, it is of interest to know how these

additional phosphorylations could affect their binding to c-Cbl

In order to understand the importance of these additional phosphorylations, we

have determined the crystal structures of human c-Cbl TKB domain in complex with

double phosphorylated Spry2 peptide and in complex with double phosphorylated

EGFR peptide From the crystal structures and the biophysical studies using surface

plasmon resonance, we observe that these peptides bind TKB with a significantly

reduced affinity However, the overall binding mode remains unchanged except few

small changes observed Chapter III of this thesis reports the details of this study

1.8 H2S signalling through p38 MAPK

It is worthy to mention that the kinase identified to be responsible for EGFR

ser1070/1071 phosphorylations is p38 MAPK (Adachi et al., 2009; Nishimura et al.,

2009) Adachi et al investigated the detailed mechanism underlying Epigallocatechin

Gallate (EGCG) induced downregulation and degradation of EGFR in SW480 colon

cancer cells They observed that EGCG required neither an ubiquitin ligase (c-Cbl)

binding to EGFR nor a phosphorylation of EGFR at tyrosine residues for EGFR

downregulation EGCG induced phosphorylation of p38 MAPK, and the inhibition of

p38 MAPK suppressed the internalization and subsequent degradation of EGFR

induced by EGCG Moreover, EGCG caused phosphorylation of EGFR at

Ser1070/1071, a site that is critical for its downregulation and this was also

suppressed by p38 MAPK inhibition Similarly, Nishimura et al showed that p38 is

responsible for phosphorylation (ser1070/1071) and endocytosis of EGFR in a

tyrosine kinase activity-independent manner

Interestingly, p38 is tightly regulated by H2S (Yang et al., 2004; Yang et al., 2006;

Yang et al., 2007; Yusof et al., 2009), which is a product of CSE In the previous

sections we have described that H2S plays multiple important roles in apoptosis,

inflammation, vasodilation, and neurotransmission to mention a few In depth studies

have revealed their connection with p38 MAPK Activation of p38 in the presence of

H2S was observed in several studies, resulting in apoptosis (Yang et al., 2004; Yang et

al., 2006; Yang et al., 2007), anti-inflammation (Yusof et al., 2009), angiogenesis

(Papapetropoulos et al., 2009), and anti-proliferation of human osteoblastic cells (Imai

et al., 2009) On the other hand, inhibition of p38 is also observed in the presence of

H2S, exhibiting anti-apoptosis (Hu et al., 2009; Rinaldi et al., 2006; Sivarajah et al.,

2009), and anti-inflammation behavior (Hu et al., 2007; Sivarajah et al., 2009) All of

these studies have demonstrated that p38 is downstream of H2S for various effects;

however the detailed mechanism is still unclear These studies together with c-Cbl

mediated EGFR endocytosis are summarized in the following schematic drawing

(Figure 20)

Figure 20 Molecular pathways involving CSE and c-Cbl H2 S, a product of CSE, is shown to regulate p38 in different studies, resulting in pro-apoptosis (Yang et al., 2004; Yang et al., 2006; Yang et al., 2007), anti-apoptosis (Hu et al., 2009; Rinaldi et al., 2006; Sivarajah et al., 2009), anti-inflammation (Hu et al., 2007; Sivarajah et al., 2009; Yusof et al., 2009) In another context, activated p38 can specifically phosphorylated EGFR Ser1070/1071 residues, causing EGFR endocytosis (Adachi et al., 2009; Nishimura et al., 2009) Cbl can also cause EGFR endocytosis by binding to activated EGFR phosphotyrosine1069 (which is adjacent to the p38 Ser1070/1071 phosphorylation site) through ubiquitination (Waterman et al., 2002; Ravid et al., 2002) Spry2 can inhibite EGFR endocytosis by sequestering c-Cbl through Spry2 phosphotyrosine 55 (Hall et al., 2003; Haglund et al., 2005; Fong et al., 2003) We speculate that the pathway in the dashed box could be a possible pathway that occurs in apoptotic cells caused by H 2 S However, this warrants further experimental verification

1.9 Chapter summary

CSE is a critical protein controlling the production of H2S which is an important

gasotransmitter physiologically H2S level dysregulation is implicated in several common diseases including Alzheimer's disease, coronary heart disease,

spontaneously hypertension, Down syndrome, diabetes mellitus, paw oedema,

pancreatitis, lung injury, haemorrhagic shock, and endotoxic shock Unfortunately, no

CSE activator is available at this moment, though covalent binding/low potency

inhibitors are available Therefore, it is important to study the structure of human CSE

towards designing inhibitor or activator of CSE for therapeutic intervention

H2S can regulate p38 activation, which in another context is responsible for

EGFR Ser1070/1071 phosphorylation and subsequent EGFR endocytosis c-Cbl,

through ubiquitination, could also mediate EGFR endocytosis by binding to EGFR

phosphotyrosine1069 Spry2 can bind to c-Cbl through its phosphotyrosine55 It is

suggested that the adjacent Threonine56 in Spry2 could also be phosphorylated,

although the function is not established As these serine/threonine phosphorylations in

Spry2 or EGFR are close to the phosphotyrosine in c-Cbl recruiting motif, it is

important to know if these additional phosphorylations might affect c-Cbl binding As

a continuation of our previous studies on c-Cbl, we crystallized c-Cbl TKB domain

with double phosphorylated EGFR or Spry2 and analyzed the effect of multiple

phosphorylation in c-Cbl recruitment though surface plasmon resonance studies