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

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 H2S, 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 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, H2S Hydrogen sulfide (or hydrogen sulphide) is a colorless, flammable, water-soluble gas with the characteristic smell of rotten eggs. For decades, H2S 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-1β (IL1β). HO1: haem oxygenase (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 H2S 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 (NaHS). NaHS is an H2S 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 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 H2S. (A) Relative carbon dioxide production and oxygen consumption of mice exposed to 80 ppm of hydrogen sulfide. (B) Core Body Temperature of mice during 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 ischemia and 72 h reperfusion. On the other hand, the hydrogen sulfide donor sodium hydrosulfide (NaHS) attenuated renal dysfunction and injury caused by 45 ischemia and 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 H2S 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, ml/kg onto kidneys) administered 15 before 45 ischemia and before h reperfusion (IRI NaHS). *Pgi|262476|gb|AAB24700.1| cystathionine gamma-lyase [Homo sapiens] MQEKDASSQGFLPHFQHFATQAIHVGQDPEQWTSRAVVPPISLSTTFKQGAPGQHSG FEYSRSGNPTRNCLEKAVAALDGAKYCLAFASGLAATVTITHLLKAGDQIICMDDVY GGTNRYFRQVASEFGLKISFVDCSKIKLLEAAITPETKLVWIETPTNPTQKVIDIEG CAHIVHKHGDIILVVDNTFMSPYFQRPLALGADISMYSATKYMNGHSDVVMGLVSVN CESLHNRLRFLQNSLGAVPSPIDCYLCNRGLKTLHVRMEKHFKNGMAVAQFLESNPW VEKVIYPGLPSHPQHELVKRQCTGCTGMVTFYIKGTLQHAEIFLKNLKLFTLAESLG GFESLAELPAIMTHASVLKNDRDVLGISDTLIRLSVGLEDEEDLLEDLDQALKAAHP PSGIHS c-Cbl protein sequence >gi|29731|emb|CAA40393.1| c-cbl [Homo sapiens] MAGNVKKSSGAGGGTGSGGSGSGGLIGLMKDAFQPHHHHHHHLSPHPPGTVDKKMVE KCWKLMDKVVRLCQNPKLALKNSPPYILDLLPDTYQHLRTILSRYEGKMETLGENEY FRVFMENLMKKTKQTISLFKEGKERMYEENSQPRRNLTKLSLIFSHMLAELKGIFPS GLFQGDTFRITKADAAEFWRKAFGEKTIVPWKSFRQALHEVHPISSGLEAMALKSTI DLTCNDYISVFEFDIFTRLFQPWSSLLRNWNSLAVTHPGYMAFLTYDEVKARLQKFI HKPGSYIFRLSCTRLGQWAIGYVTADGNILQTIPHNKPLFQALIDGFREGFYLFPDG RNQNPDLTGLCEPTPQDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHL MCTSCLTSWQESEGQGCPFCRCEIKGTEPIVVDPFDPRGSGSLLRQGAEGAPSPNYD DDDDERADDTLFMMKELAGAKVERPPSPFSMAPQASLPPVPPRLDLLPQRVCVPSSA SALGTASKAASGSLHKDKPLPVPPTLRDLPPPPPPDRPYSVGAESRPQRRPLPCTPG DCPSRDKLPPVPSSRLGDSWLPRPIPKVPVSAPSSSDPWTGRELTNRHSLPFSLPSQ MEPRPDVPRLGSTFSLDTSMSMNSSPLVGPECDHPKIKPSSSANAIYSLAARPLPVP KLPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSSRACDCDQQIDSCTYEAMYNIQSQA PSITESSTFGEGNLAAAHANTGPEESENEDDGYDVPKPPVPAVLARRTLSDISNASS SFGWLSLDGDPTTNVTEGSQVPERPPKPFPRRINSERKAGSCQQGSGPAASAATASP QLSSEIENLMSQGYSYQDIQKALVIAQNNIEMAKNILREFVSISSPAHVAT 134 135 136 137 138 [...]... 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 20 sometimes associated with mental retardation (Tang et al., 2006; Wang and Hegele, 2003) The PLP content in the two natural CSE. .. 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 H2S, pyruvate and ammonia (adapted from Huang et al., 2010) 1.4.3 CSE regulation in vivo CSE is phosphorylated upon DNA damage, probably... disease, diabetes and cancer (Bachmaier et al., 2000) 32 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... 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... (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... 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... 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... 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 19 6-ethyl-chenodeoxycholic acid (6E-CDCA), a synthetic FXR ligand, to rats protected increased CSE expression level, increased H2S generation, reduced... 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... phosphotyrosine containing motifs 26 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 . 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. 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. 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