S-adenosylmethionine synthesis: Molecular mechanisms and clinical implications

Phannacol. Ther. Vol. 73, No. 3, pp. 265-280, 1997 Copyright 0 1997 Elsevier Science Inc.

ISSN 0163-7258197 $32.00 PI1 sO163-7258(96)00197-O

ELSEVIER

Associate Editor: P. K. Chiang

S-Adenosylmethionine Synthesis: Molecular Mechanisms and Clinical Implications

Jose’ M . Mato, * Luis Alvarez, Pablo Ortis, and Ma& A. Pajares INSTITUTO DEINVESTIGACIONES BIOM~DICAS,CSIC,ARTURODUPERIER4,28029-MADRID,SPAIN

ABSTRACT. Methionine adenosyltransferase (MAT) is an ubiquitous enzyme that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. In mammals, there are two genes coding for MAT, one expressed exclusively in the liver and a second enzyme present in all tissues. Molecular studies indicate that liver MAT exists in two forms: as a homodimer and as a homotetramer of the same oligomeric subunit. The liver-specific isoenzymes are inhibited in human liver cirrhosis, and this is the cause of the abnormal metabolism of methionine in these subjects. PHARMACOL. THER. 73(3):265-280,1997. 0 1997 Elsevier Science Inc.

KEY WORDS. S-adenosvlmethionine, S-adenosylhomocysteine, methionine adenosyltransferase, liver disease, oxidative stress, glutathione.

CONTENTS

1. INTRODUCTION. . . . . . . . . 2. METHIONINEMETABOLISM. . .

2.1. THEMETHYLATIONCYCLE 2.2. THEMETHYLATIONCYCLE

IN LIVERDISEASE . . . . . 2.3. S-ADENOSYLMETHIONINE

...... 265 3.2.

...... 266

...... 266 3.3.

...... 267

PROTECTIONOFHEPATIC INJURY . .270 3.4. 3. MOLECULARBIOLOGYOF

S-ADENOSYLMETHIONINESYNTHESIS. . .272

HEPATIC METHIONINE ADENOWLTRANSFERASE . . . . . . .274 REGULATIONANDSTRUCT~REOF THELIVER-SPECIFICMETHIONINE ADENOSyLTRANSFERASEGENE . . . .274

3.1. DIFFERENTFORMSOFMETHIONINE AcKNowLEDGEMENTs.............~~~

ADENOSYLTRANSFERASE REFERENCES . . . . . . . . . . . . . . . . . .275 INMAMMALS . . . . . . . . . . ...272

STRUCTURE oFmE LIVER-SPECIFICMETHIONINE ADENOSyLTRANSFERASE . . . . . . .273 GENETICDEFICIENCY IN

ABBREVIATIONS. AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; BSO, buthionine sulfoximine; DMSO, dimethylsulfoxide; GSH, glutathione; GRE, glucocorticoid responsive element; GSSG, oxidized glutathione; HIV, human immunodeficiency virus; MAT, methionine adenosyltransferase.

1. INTRODUCTION

The methylation cycle (Fig. 1) involves the conversion of methionine, via S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy), into homocysteine, followed by reconversion of homocysteine into methionine. This cycle has three major cellular functions. First, it provides

AdoMet, necessary for polyamine synthesis and for the methylation of numerous essential cell constituents (collectively referred to as “Acceptor” in Fig. l), such as phospholipids,

methyl-accepting proteins, CpG islands in DNA, adrener- gic, dopaminergic and serotoninergic molecules. Second, it feeds the transsulphuration pathway that leads to the for-

mation from homocysteine of glutathione (GSH), the main cellular antioxidant, required for the detoxication of vari-

ous compounds and for the scavenging of free radicals. Finally, it recycles 5-methyltetrahydrofolate into tetrahydrofolate, a necessary cofactor for the synthesis of DNA and RNA.

Several conditions and compounds are known to markedly impair one or several steps of the methylation cycle.

*Corresponding author.

Various chronic liver disorders, including alcoholic, biliary

and viral cirrhosis, markedly decrease the hepatic activities of methionine adenosyltransferase (MAT; ATP:L-methi-

onine S-adenosyltransferase, EC 2.5.1.6), the enzyme that catalyzes the synthesis of AdoMet from L-methionine and ATP, and of phospholipid N-methyltransferase, the enzyme that catalyzes the synthesis of phosphatidylcholine from

phosphatidylethanolamine by three successive N-methylations of the free amino group of ethanolamine (Martin-Duce et

al., 1988). Numerous drugs, particularly anticancer and antiviral agents (Chiang et al., 1977; Cantoni and Chiang,

1980; De Clercq, 1987), inhibit AdoHcy hydrolase, the enzyme that catalyzes the hydrolysis of AdoHcy into its com-

ponents, homocysteine and adenosine, which probably account for their mechanism of action. The activity of methionine synthase, the enzyme that catalyzes the methylation of homocysteine to form methionine, is impaired by vitamin B,, or folate deficiency (B,, is an essential cofactor for the regeneration of tetrahydrofolate), or by inactivation induced by the administration of the anaesthetic nitrous oxide or ethanol ingestion (Scott et al., 1981; Weir et al., 1988; Trimble et al., 1993). Finally, several hepatotoxins (including alcohol, carbon tetrachloride, galactosamine

266 J. M. Mato et al.

and acetaminophen), which are detoxified by GSH, pro- voke the depletion of this reductive agent, which, in turn, inactivates liver MAT (Pajares et al., 199210).

It is obvious from the above results that the methylation cycle plays an essential role in maintaining cell function

and structure, and that its metabolic importance hardly can be overestimated. AdoMet plays a central role in the meth-

ylation cycle by controlling both the remethylation of homocysteine to methionine and its catabolism through the

transsulfuration pathway. Moreover, AdoMet participates in probably as many cellular reactions as ATP, and, therefore, it is not surprising that a normal adult makes about 8 g of

AdoMet per day, the majority of it in the liver, where it is also mainly consumed (Mudd and Poole, 1975; Mudd et al., 1980). Accordingly, the synthesis and utilization of AdoMet, espe-

cially by the liver, needs to be carefully controlled. This review focuses on the regulation of the methylation

cycle and its alteration during liver dysfunction. Recent studies strongly suggest that AdoMet therapy corrects im- pairments of the methylation cycle during liver dysfunc-

tion. Finally, we will review our current information on the

regulation of MAT gene expression and MAT structure.

2. METHIONINE METABOLISM 2.1. The Methrlation Cycle

In mammals, dietary methionine is converted by MAT into

AdoMet (Mudd and Cantoni, 1958; Cantoni, 1975) (Fig. 1). This is the only operating reaction to catabolize me-

thionine, since transamination of this essential amino acid

does not occur under normal metabolic conditions (Wu and Thompson, 1989; Cooper, 1989). The majority of methionine is metabolized by the liver, which agrees with this tissue having the highest specific activity of MAT (Finkel-

stein, 1990). AdoMet serves as the donor of methyl groups in over 100 methylation reactions, e.g., phospholipid,

DNA, RNA and protein methylation, which are essential for cellular structure and function. Moreover, AdoMet is the

methyl donor for all detoxifying methylation reactions (thi-

Tetra-

tetrahvdrofo,ate 7 MethioniniAdcTines

5-Methyl-

Homocysteine k Acceotor

.%phu/ \ /I ’ amino acids,

Methylated acceptor

GSH AdoHcy

FIGURE 1. The methylation cycle involves the conversion of methionine, via AdoMet and AdoHcy, into homocysteine, followed by reconversion of this amino acid into methionine. See the text for a detail description of the cycle and its functions.

ols, histamine, etc.). Whereas a specific enzyme catalyzes each of these reactions, the common product to all methylation reactions is AdoHcy (Fig. 1). Most AdoMet-dependent methylation reactions are strongly inhibited in vitro by AdoHcy and, therefore, might be controlled in viva by the ratio AdoMet/AdoHcy.

Transfer of the propylamine group of AdoMet for the synthesis of polyamines is also an important biological

function of this molecule (Tabor et al., 1958) (Fig. 1). Un- der normal conditions, this pathway does not account for more than 5% of the available AdoMet, but this percent can be increased under conditions of increased polyamine

synthesis, as during liver regeneration (Mudd and Poole, 1975; Mudd et al., 1980; Ubagai et al., 1994).

Of all the enzymes that use AdoMet in the liver, glycine

N-methyltransferase, a folate binding protein, is the most abundant and accounts for about 1% of the hepatic cytoso-

lit protein (Cook and Wagner, 1984). This enzyme, which is mainly expressed in the liver, catalyzes the synthesis of

sarcosine from AdoMet and glycine, and, in turn, is rapidly converted again to glycine. The function of this enzyme, which is inhibited by 5-methyltetrahydrofolate, but is not

inhibited by AdoMet (Cook and Wagner, 1984), is to re- move excess methyl groups, and thus, it acts to regulate the AdoMet/AdoHcy ratio, even under conditions where the supply of AdoMet is limited (Mato et al., 1994). AdoHcy concentration is maintained at low levels by being further

converted into adenosine and homocysteine by AdoHcy hydrolase (Fig. 1). AdoHcy hydrolase is a reversible en-

zyme, which thermodynamically favours the synthetic direction. Consequently, conditions that lead to homocys-

teine accumulation result in a decreased AdoMet/AdoHcy ratio and inhibition of methylation reactions. Therefore, to

operate the methylation cycle in viva, homocysteine and adenosine have to be removed rapidly. Homocysteine is converted into either cystathionine and its derivatives (cysteine, GSH, taurine, inorganic sulphate) by the transsulfuration pathway, or used again for the resynthesis of methionine (Fig. 1).

Whereas the latter are the main two metabolic pathways to dispose of homocysteine, this amino acid may also be protein-bound and its excess may be released into the ex-

tracellular medium. Increased plasma levels of this amino acid have been shown to be a risk factor for the development of cardiovascular disease (Clarke et al., 199 1; Miller et

al., 1994; Selhub et al., 1995). Homocysteine methylation to form methionine can be

achieved by two different reactions, methionine synthase (also called methylfolate-homocysteine methyltransferase) and betaine-homocysteine methyltransferase (Fig. 1). Be- taine-homocysteine methyltransferase uses betaine, a metabolite of choline, as the methyl donor for homocysteine methylation. Consequently, homocysteine methylation is necessary for both folate cycling and, therefore, DNA and

.RNA synthesis and choline metabolism. Whereas methionine synthase occurs in all mammalian tissues, betaine-homocysteine methyltransferase is mainly present in the liver.

Synthesis of S-adenosylmethionine 267

The first enzyme in the transsulfuration pathway is cys-

tathionine P-synthase, which requires vitamin B, as a co-

factor. This enzyme uses homocysteine and serine to form cystathionine, and is the third enzyme that competes for homocysteine in the liver (Fig. 1). y-Cystathionase, another vitamin Bh-dependent enzyme, converts cystathionine into cysteine and a-ketobutyrate, and is also mostly present in the liver. Cysteine is the precursor for the synthesis of a number of important metabolites, including

GSH, taurine and sulphate. Glutathione is synthesized exclusively intracellularly in

its reduced form (GSH). The liver is the major organ for

GSH synthesis and excretion into circulation and bile. This thiol is the predominant low molecular cellular antioxidant and functions as a multipotential metabolite. GSH is trans-

located from the cytosol, where its synthesis takes place, to mitochondria and nucleus, and serves as an antioxidant re-

ducer and for detoxifying electrophilic compounds. Among other functions, GSH participates in reductive processes that are essential for the synthesis and degradation of proteins, protection of cells against reactive oxygen compounds and free radicals, and regulation of the redox state of a number of biological macromolecules. GSH can also

act as a coenzyme for several enzymatic reactions, and is considered to be a storage and transport form of cysteine. Extracellular GSH is hydrolyzed on the outer surface of

cells, and the constituent amino acids, including cysteine, enter cells and are used for GSH synthesis (Meister and

Anderson, 1983). GSH depletion is associated with a reduction in the number of mitochondria, mitochondrial swelling and degeneration with rupture of membrane archi-

tecture and increased density (Martensson and Meister, 1989; Martensson et al., 1989; Corrales et al., 1991).

Before reviewing how the methylation cycle is regulated, it is important to note that the cycle, as it is shown in Fig. 1, only occurs in the liver, since most other tissues lack one or more of the enzymes depicted in the diagram. All tissues

can make and utilize AdoMet, although the specific activ-

ity of MAT can vary more than loo-fold from one tissue to another, being maximal in the liver. A number of tissues, the liver, small intestine, kidney, pancreas, brain and adi- pose tissue, can make cystathionine, although, with the ex-

ception of the liver, most of them either do not contain cystathionase or express it in low amounts (Finkelstein, 1990). Consequently, with the exception of the liver, the transsulfuration pathway is either absent or of minor importance in mammalian tissues. Thus, in these tissues, cysteine is an es-

sential amino acid that is supplied either through the diet or through the hepatic synthesis of this amino acid and its subsequent excretion in the form of GSH.

As previously mentioned, in mammals, the liver is the main place for methionine metabolism. Up to 85% of all

methylation reactions and as much as 48% of methionine metabolism occurs in the liver (Mudd and Poole, 1975). In the rat liver, the half-life of the methyl group of methionine has been estimated to be about 5.5 min and the fraction of homocysteine converted to cystathionine during each cycle

is 45% (Finkelstein, 1990). In humans, the fraction of

available homocysteine converted to cystathionine during

each cycle has been estimated to be 53%, and this percent could drop to about 20% when the dietary content of methyl groups was restricted (Mudd and Poole, 1975), as a way to preserve methionine consumption. In humans, the estimated

half-life of hepatic AdoMet is 2.4-5.9 min under normal dietary conditions, and a normal adult was found to make approximately 6-8 g of AdoMet per day.

A central role of AdoMet in the regulation of methionine metabolism was proposed by Finkelstein (Finkelstein and Martin, 1984a,b; Finkelstein et al., 1975a), based on his

work with cystathionine P-synthase and betaine-homocysteine methyltransferase, and with the work of Stokstad

(Kutzbach and Stokstad, 1967, 1971) with methionine syn-

thase. It has been shown that AdoMet reduces the synthesis of 5-methyltetrahydrofolate, inhibits betaine-homocysteine methyltransferase, and increases the synthesis of cystathionine by activating cystathionine P-synthase. Conse-

quently, intracellular AdoMet concentration is an important factor to modulate the distribution of homocysteine between remethylation and cystathionine synthesis. When the concentration of AdoMet increases, for instance, as a

result of an elevation of plasma methionine, the synthesis of methionine by methylation of homocysteine is reduced and the utilization of this amino acid through the transsul-

phuration pathway is enhanced.

2.2. The Methylation Cycle in Liver Disease

Alcoholic liver disease is the most common clinical com- plication of alcoholism, and accounts for a majority of the cases of cirrhosis of the liver in the industrialized countries.

Ethanol-induced chronic liver disease is associated with alterations of the methionine cycle, and it has been suggested that the increased cancer risk associated with chronic alcoholism might be related to these changes.

The relationship between alcohol and methionine me-

tabolism has caught the attention of researchers for about 50 years. Already in 1947, Kinsell and co-workers first de-

scribed both the increased serum methionine and the decreased clearance of this amino acid in patients with alcoholic cirrhosis. Almost 40 years after Kinsell’s original

observation, Chawla and colleagues reported that the abnormal clearance of methionine observed in human liver cirrhosis was associated with decreased levels of cysteine and its derivatives, although no accumulation of any metabolite between methionine and cysteine was observed in

those experiments (Horowitz et al., 1981; Chawla et al., 1984). Following an oral load of methionine in cirrhotic patients, only 38% of the amino acid was excreted as uri-

nary sulfate within 24 hr in comparison with 71% in controls with normal liver function. Based on these results, the

authors proposed that the basic defect was a reduction in the capacity of the liver to synthesize AdoMet.

In 1988, this hypothesis appeared to be correct by the showing of a marked deficiency (about 50%) of MAT ac-


tivity in liver biopsies from patients with alcoholic hepatic cirrhosis as compared with samples from subjects with normal liver function (Martin-Duce et al., 1988; Cabrero et al., 1988). Moreover, liver biopsies from patients with viral cirrhosis showed a similar reduction of hepatic MAT (Martin- Duce et al., 1988). Reduced hepatic MAT activity has also been reported in liver biopsies from patients with primary biliary cirrhosis and Wilson’s disease (Gaul1 et al., 1981). This data clearly indicates that abnormal methionine metabolism is associated with liver dysfunction and is inde-

pendent of the etiology of the disease. Moreover, methio-

nine clearance deficiency correlates with the severity of liver disease in cirrhotic patients (Marchesini et al., 1992).

Methionine metabolism has also been investigated in various experimental models of liver disease. The most

thorough studies have been carried out using rats chronically intoxicated with either ethanol or Ccl,. Limited in-

formation on the effect of chronic ethanol feeding to baboons on liver methionine metabolism is also available. Ccl+ a toxic agent commonly used for the study of liver injury in different experimental animal models, is a substrate

for P450, where it is converted to Ccl, radicals, which generate Ccl,00 radicals by reacting with molecular oxygen. Since Ccl3 radicals react with membranes and induce lipid

peroxidation, membrane damage by free radical chain reac-

tion has been proposed as the major cause of hepatocellular injury by Ccl,, the initial event being probably mitochon-

drial membrane injury (Butler, 1961; Lai et al., 1979; Slater, 1984; Yasuda et al., 1980). Similarly, microsomes from ethanol-fed rats have been shown to generate reactive oxygen intermediates, such as superoxide and hydroxyl radicals, at elevated rates compared with controls. This is associated with an increased lipid peroxidation in ethanol-fed

animals, and furthermore, there is also evidence for ethanol-associated lipid peroxidation in humans (Lieber and

Leo, 1992). Ethanol- and CC&nduced liver injury are associated with

a reduction in hepatic AdoMet and an increase in AdoHcy,

which, in turn, lead to a reduction in the AdoMet/AdoHcy ratio (Barak et al., 1987; Trimble et al., 1993; Varela-Mor- eiras et al., 1995). In rats with normal liver function, the hepatic AdoMet/AdoHcy ratio was shown to be between 3 and 4. After Ccl, administration or ethanol feeding, this

ratio reached the value of 1. This was due both to a reduction in the hepatic concentration of AdoMet and to an increase in the levels of AdoHcy. In baboons, the concentration of AdoHcy was not determined after ethanol feeding (Lieber et al., 1990). However, since the reduction of hepatic AdoMet was clearly significant, one may assume that

a reduction in the AdoMet/AdoHcy ratio might also take place as a result of ethanol consumption in this experimental model.

The effect of this reduction in the hepatic concentration of AdoMet and/or in the AdoMet/AdoHcy ratio on cellular methylation reactions has been studied by measuring DNA-methylation in uiuo. A marked hypomethylation of hepatic DNA (about 2-fold) has been observed in liver

samples from rats treated for 3 weeks with Ccl,, as compared with control animals (Varela-Moreiras et al., 1995).

Because of this reduction in the concentration of AdoMet and in the AdoMet/AdoHcy ratio observed during liver injury, other methylation reactions, besides DNA methylation, might also be inhibited in response to these agents; however, no direct evidence at this respect is available yet.

Phospholipid N-methyltransferase was first found to be markedly deficient in liver biopsies from patients with alco-

holic or viral cirrhosis (Martin-Duce et al., 1988). It is important to note that this deficiency in hepatic phospholipid

N-methyltransferase is independent of the low availability of AdoMet and of the AdoMet/AdoHcy ratio in liver cir-

rhosis, since the activity of this enzyme was determined in vitro using washed microsomal membranes and radioactive

AdoMet, and the reaction followed by measuring the incor- poration of labeled methyl groups into phospholipids (Mar- tin-Duce et al., 1988). A similar reduction in hepatic phospholipid N-methyltransferase has also been observed in baboons chronically fed ethanol (Lieber et al., 1994a) and

in hypoxic rat liver (Chawla and Jones, 1994). Since methylation of phosphatidylethanolamine into phosphatidylcholine increases the fluidity of membranes (Mato and Ale-

many, 1983), these findings might be related to the lower liver plasma membrane fluidity observed in rats chronically

fed ethanol (Schuller et al., 1986), and hence, to the decreased bile flow in chronic liver disease. In the baboon

model, the administration of phosphatidylcholine has been shown to protect against ethanol-induced fibrosis and cirrhosis (Lieber et al., 199413). Based on these results, the potency of polyenyl-phosphatidylcholine to restore liver functions and correct various states of liver pathology is currently being evaluated in an ongoing clinical trial.

In eukaryotic cells, the expression of genetic information is often associated with the extent and pattern of DNA meth-

ylation (Razin and Riggs, 1980; Tilghman, 1993), and it has been proposed that permanent alterations in gene expression might result if more than one cycle of DNA replica-

tion occurs under conditions that favour hypomethylation (Dizik et al., 1991). Deficient methylation of certain genes is considered to play a pivotal role in the development of hyperplasia and carcinoma. The carcinogenic effect of ethionine, a methionine analog that is converted by MAT

into S-adenosylethionine, most probably is explained by the inhibitory effect of the later compound on the methylation of cytosine residues of DNA. This methylation is

known to be involved in repressing the expression of certain genes, including oncogenes. Diets containing limiting amounts of lipotropes, such as methionine, choline, folic

acid, and vitamin B12, reportedly promote chemical carcinogenesis. In addition, chronic administration of methyl- deficient diets causes spontaneous hepatoma in laboratory rats without any exogenous carcinogen (Wainfan et al., 1991). These animals have subnormal hepatic AdoMet and hypomethylated DNA (Newbeme and Rogers, 1986). These results suggest that the reduction in AdoMet and/or the AdoMet/AdoHcy ratio and the concomitant DNA hy-

Synthesis of S-adenosylmerhionine 269

pomethylation that occurs during liver injury might be cru-

cial factors in the development of carcinoma.

In Chinese hamster ovary cells transfected with rat liver MAT, the concentration of ATP was markedly diminished, as compared with wild-type cells (Sanchez-Gongora et al., 1996). Based on these results, we propose that the reduction in MAT activity observed in liver disease might be an adaptive mechanism to spare ATP to combat oxidant-induced

cell injury and prevent mitochondrial de-energization. In this context, the reduction in MAT observed during liver in-

jury might have two types of effects: one beneficial, to spare ATP, and the other detrimental, reduced methyfation of DNA and other molecules, reduced folate cycling and GSH synthesis. In the short term, a reduction in the consumption of ATP might prevent the cell from going into irreversible cell damage, whereas the effects on the methylation and transsulfuration pathways, if transient, might be

reversible. However, a sustained inhibition of the methylation cycle, as that seen in chronic hepatic disease, would

also lead to irreversible liver damage. This agrees with the finding that the administration of diets containing limiting

amounts of lipotropes, such as methionine and choline, reportedly promote hepatic steatosis resembling the fatty liver of alcoholic steatosis (Lieber et al., 1965). In addition,

as previously mentioned, chronic administration of methyl-

deficient diets causes spontaneous hepatoma in laboratory rats without any exogenous carcinogen.

Since AdoHcy hydrolase is a reversible enzyme, increases

in hepatic AdoHcy should be accompanied by increases in the cellular content and/or the serum levels of homocys-

teine. This hypothesis has proven to be correct by showing that in rats treated with CC&, during 3 weeks the serum

levels of homocysteine increased more than 20-fold (Varela- Moreiras et al., 1995).

Folic acid has been shown to be markedly reduced in liver specimens from cirrhotic patients, as well as in experi-

mental models of liver disease (Chanarin et al., 1985; Mc- Cormick and Munro, 1994; Varela-Moreiras et al., 1995). It has been proposed that in liver disease, folate deficiency probably is caused by impaired uptake by the intestine, as

well as decreased storage in the damaged liver, and not by dietary insufficiency (Chanarin et al., 1985; McCormick

and Munro, 1994). Since folate deficiency has been shown to induce homocysteinemia in rats, the assumption has been made that the primary effect of ethanol or Ccl, is a

failure of the liver to methylate homocysteine to form methionine. In keeping with this hypothesis, it has been reported that alcohol administration to rats results in de-

creased methionine synthase activity (Trimble et al., 1993). As previously mentioned, liver cirrhosis is associated

with an impaired hepatic synthesis of GSH and defective formation of AdoMet (Mato et al., 1994). After an oral methionine load, the plasma levels of both cysteine and GSH increase markedly in control subjects with normal liver function, but in cirrhotic patients, only minor changes in the plasma levels of both molecules were observed after the administration of this amino acid (Horowitz et al., 1981).

We have shown that the defective formation of AdoMet in

liver disease is not associated with a reduced expression of

the MAT gene, but that the impaired synthesis of GSH and the reduced synthesis of AdoMet are both acting together in a self-perpetuating cycle, where a reduction in hepatic GSH leads to an inhibition of MAT. This conclusion is

mainly based on two observations. First, the administration to rats of buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) leads both to a reduction in the hepatic content of GSH and to an inhibition of MAT (Corrales et

al., 1991). Second, the administration of GSH-ethyl ester

(a permeable derivative of GSH, which is hydrolysed intracellularly to provide GSH and ethanol) together with BSO, prevents both the reduction in hepatic GSH levels and the inhibition of MAT activity (Corrales et al., 1991). BSO, like Ccl, or ethanol, did not affect the hepatic expression of the MAT gene (Corrales et al., 1992; Alvarez et al.,

1993). The mechanism behind these experiments seems to in-

volve the inhibition of liver MAT by free radicals (OH, NO) and other agents (oxidized glutathione, GSSG) gener-

ated during oxidative stress, and the protection of this process by the antioxidant and free radical scavenger GSH

(Mato et al., 1994). According to this model, a reduction of hepatic GSH would leave MAT vulnerable to the inhibi-

tory action of free radicals and other oxidant agents. In turn, the inhibition of MAT by these agents would lead to a decrease in GSH synthesis and an even greater reduction of MAT activity. Redox regulation of MAT activity is supported

by the following results. First, several thiol-reacting reagents inhibit purified rat liver MAT (Berger, 1985; Pajares et al., 1991; Avila et al., 1996). Second, purified rat liver MAT is

inhibited by GSSG, a process that is inhibited by GSH (Pa- jares et al., 199213). Third, NO is a potent inhibitor of he-

patic MAT both in viva and in vitro (Avila et al., 1996). Interruption of the methylation cycle has been docu-

mented in a variety of clinical and experimental conditions, in addition to those of ethanol and Ccl, commented above. These conditions include human immunodeficiency virus (HIV) infection, tumor necrosis factor-a, and hy-

poxia. Cerebral spinal fluid from HIV-infected children (Surtees et al., 1990) and adults (Keating et al., 1991) has

been shown to contain decreased levels of AdoMet, and an impairment in methylation reactions has been proposed to be responsible for the vacuolar myelopathy present in some

patients infected with HIV (Keating et al., 1991). In a cell culture system using L929 cells, it was found that the cytotoxicity of tumor necrosis factor-a was markedly enhanced

by elevating AdoHcy by the addition of adenosine and homocysteine, which push AdoHcy hydrolase in the synthetic direction (Bergmann et al., 1994). In HIV-infected patients, cerebral spinal fluid GSH and cysteinyl-glycine were also found to be diminished (Castagna et al., 1995). More- over, several groups have demonstrated a reduction in GSH in several tissues from HIV-infected patients (Eck et al., 1989; Buhl et al., 1989; Staal et al., 1992). GSH deficiency may potentiate HIV replication and accelerate the progres-

270

sion of the disease (Staal et al., 1992). Moreover, decreased GSH levels have been proposed to play a role in the etiology of several neurological disorders such as Parkinson’s disease (Di Monte et al., 1992).

Hypoxia has also been implicated in the interruption of

the methylation cycle. Chawla and Jones (1994) have

shown a reduction of the expression of the hepatic MAT gene and diminished enzymatic activity in hypoxia in rats.

Several lines of evidence suggest that superoxide radicals and related species are generated in the liver during hyd poxia. As mentioned above, these reactive oxygen species might be responsible of the inhibition of MAT. As during cell injury induced by oxidative stress, hypoxia produces a depletion of intracellular ATP, and the reduction in he-

patic MAT activity, therefore, might be a protective mechanism to spare cellular ATP.

2.3. S-Adenosylmethionine Protection of Hepatic Injury

The above results prompted numerous laboratories to study

the efficacy of AdoMet in different experimental models of liver cell injury. This was only possible after a stable salt of

AdoMet was manufactured by BioResearch S.p.A. (Milano, Italy) in 1977. Th ree different models have mainly been used: liver necrosis caused by the metabolism of chemicals (Ccl,, acetaminophen, ethanol, galactosamine), ischemia- reperfusion cell injury, and estrogen-induced cholestasis.

Ccl, and acetaminophen are two well-studied hepatotoxins. The metabolism of these two hepatotoxic chemicals

by the P450 mixed function oxidase system of the endoplas- mic reticulum leads to irreversible cell injury through mem-

brane damage (primary mitochondrial membrane damage) as a result of peroxidation of the constituent phospholipids.

Lipid peroxidation is initiated by either the formation of Ccl, radicals (in the case of Ccl,) or by activated oxygen species formed during the metabolism of acetaminophen. In both cases, the metabolism of the hepatotoxic agent results in GSH depletion, which, in turn, diminishes the antioxi-

dant defenses of the cell. Intraperitoneal injection of rats with Ccl, can lead to

liver cirrhosis in several weeks. In this model, the accumulation of procollagen Type 1 mRNA and collagen, and the increased expression of prolyl-hydroxylase and lipid peroxi-

dation could be ameliorated by the administration of AdoMet (Corrales et al., 1992; Gas& et al., 1996). The number of rats that developed liver cirrhosis in the Ccl, group was also higher than (50%) those in the group treated with AdoMet (15%) (Corrales et al., 1992). AdoMet administration was also shown to blunt the increases in serum homocysteine levels, the hepatic ratio of AdoMet/AdoHcy induced by Ccl,, and also normalize DNA methylation and prevent the depletion of liver folates and GSH induced by the hepatotoxic agent (Cor- rales et al., 1992; Varela-Moreiras et al., 1995). Moreover, the addition of AdoMet to monolayers of fibroblasts reduces the synthesis of collagen without having toxic effects on the cells (Casini et al., 1989).

J. M. Mato et al.

In a rat model of liver hepatotoxicity induced by galactosamine, the administration of AdoMet also improved survival (Stramentinoli et al., 1978). Galactosamine is known to induce a reduction of liver MAT activity and a depletion

of AdoMet and GSH levels (Stramentinoli et al., 1978; Ca- brero et al., 1988; Wu et al., 1996). AdoMet administration

has also been shown to improve survival in a mouse model of intoxication induced by the administration of acetamin-

ophen (Stramentinoli et al., 1979; Bray et al., 1992). AdoMet also improved the GSH depleting effect of a vari-

ety of drugs, such as acetaminophen, methadone, heroine and ethanol, in human cultured hepatocytes (Ponsoda et

al., 1991). Ethanol-induced liver injury is also ameliorated by the

administration of AdoMet. This has been demonstrated in

two different animal models, baboons and rats. Baboons were fed ethanol for 15-18 months with or without

AdoMet supplementation. AdoMet partially prevented the

hepatotoxic effects of ethanol on mitochondrial swelling, GSH and AdoMet depletion (Lieber et al., 1990). Simi- larly, in rats fed ethanol, AdoMet protected the damaging

effect of this hepatotoxin on mitochondrial function, GSH content, production of acetaldehyde and deposition of fat in the liver (Feo et al., 1986; Pascale et al., 1989; Garcia-

Ruiz et al., 1995). AdoMet administration increased hepatic GSH levels in patients with alcoholic and nonalco- holic liver disease (Vendemiale et al., 1989), and decreased

blood levels of ethanol and acetaldehyde in healthy volun- teers after the administration of ethanol (Di Padova et al.,

1984). Finally, AdoMet has been shown to antagonize the hepato-

toxic effect of cyclosporin A (Femandez et al., 1995), cyto- kines (Vara et $., 1994) and thioacetamide (Mesa et al., 1996),

and is also capable of preventing mortality induced by both acute and chronic lead intoxication (Paredes et al., 1985).

Ischemic cell injury followed by reperfusion is one of the

most important causes of coagulative necrosis in human disease. Some events occur during the period of ischemia

that result in an overproduction of toxic oxygen species (superoxide ion, hydrogen peroxide and hydroxyl radicals) on the later restoration of the oxygen during reperfusion.

Interruption of the methylation cycle induced by hypoxia may underlie the impairment of hepatic function described

in isolated perfused rat livers using a model of transplantation ischemia-reperfusion. In this model, the deterioration in hemodynamic, metabolic and biochemical indices of liver function could be reversed by administration of AdoMet (Dunne et al., 1994).

Intrahepatic cholestasis of pregnancy is a familial disor- der, characterized by pruritus and cholestatic jaundice, that usually occurs in the last trimester of each pregnancy and promptly disappears after delivery. Maternal health is unaf- fected by the disease, but the effects on the fetus are often grave and include fetal distress, stillbirth, prematurity and intracranial hemorrhage during delivery. Interruption of the methylation cycle induced by the increase in gonadal and placental hormones during pregnancy is likely to occur


and be associated to cholestasis in susceptible women. In several studies using a model of estrogen-induced cholesta-

sis in rats, bile flow impairment was shown to be prevented

by the administration of AdoMet (Stramentinoli et al., 1981; Di Padova et al., 1985). Similarly, the cholestatic ef-

fect of a variety of drugs (a-naphthyl-isothiocyanate, thioacetamide, cyclosporin A) is prevented by AdoMet treatment (Di Padova et al., 1985; Osada et al., 1986; Fernandez et al., 1995). Not only drug-induced cholestasis, but also the cholestatic syndrome induced by parenteral nutrition (Belli et al., 1994) and exhaustive exercise (Villa et al.,

1993) was prevented by AdoMet administration. Finally, in a number of clinical trials, the efficacy of AdoMet in pa-

tients suffering from cholestasis of pregnancy was also demonstrated (Frezza et aI., 1988).

After these encouraging results obtained with AdoMet in the treatment of cholestasis of pregnancy, a series of clinical trials were performed in patients suffering from cholestasis induced by chronic liver disease (Frezza et al., 1990; Giu- dici et al., 1992). The total number of patients treated in

these trials was approximately 1000. AdoMet was significantly better than placebo in the percentage of patients that improved by 50% their basal score of pruritus, fatigue,

total and conjugated bilirubin, and alkaline phosphatase. Side effects in all these trials were only minor, and no drop- outs for secondary events were reported. Return to basal

values of the parameters ameliorated by AdoMet administration was observed after withdrawal of the treatment.

Since the first demonstration of AdoMet in protecting against hepatic injury, the question has been raised whether this molecule is transported inside the liver cells. Four types

of experiments suggest that this might be the case. First, AdoMet treatment restored intracellular AdoMet concentrations in damaged hepatocytes (Traver et al., 1984), as well as in livers of animals treated with certain hepatotoxins (Stramentinoli et al., 1978; Lieber et al., 1990; Varela-

Moreiras et al., 1995). Second, in the experiments carried

out by Lieber and colleagues (1990), an enantiomer of AdoMet, which was a contaminant in their preparation,

was detected in the liver of the treated animals. Third, the administration of large doses of AdoMet by intraperitoneal injection to rats with normal liver function has been shown

to increase the hepatic content of AdoMet and AdoHcy (Finkelstein, 1990). Fourth, one individual positively diag nosed with reduced hepatic MAT deficiency and who developed abnormal neurological symptoms and neural demyelination, responded favorably to AdoMet therapy (Surtees

et al., 1991). Moreover, clear evidence about the presence of a specific permease for AdoMet has been presented in Saccharomyces cereuisiae, for which strains with disruption of the two genes encoding MAT exhibit auxotropy for AdoMet (Thomas and Surdin-Kerjan, 1991). If yeast is able to concentrate exogenously added AdoMet, the theory that this property has been maintained in mammals cannot be

criticised as extraneous. Finally, it is important to note that in addition to its intracellular effect, the available evidence does not permit elimination of the possibility of an extra-

cellular action of AdoMet in mammals when administered

exogenously. In fact, Tredger and associates (Dunne et al.,

1994) have studied the potency of AdoMet for reducing is-

chaemia/reperfusion injuries, and demonstrated that in addition to its antioxidant effect, the efficacy of the compound may also involve interactions with Type Pi purinergic receptors. Methylation of membrane phospholipids in intact cells by incubation with AdoMet has been reported in a vari-

ety of systems (Mato and Alemany, 1983). A second question has arisen regarding how this mole-

cule exerts its protective action. If AdoMet is transported inside the injured liver cells, we must assume that it will be

metabolized just as endogenous AdoMet is. The uptake of AdoMet by the hepatocytes will cause an increase in the

AdoMet/AdoHcy ratio. This may lead to an increased for-

mation of GSH, which, in turn, will facilitate the scavenging of free radicals generated by the hepatotoxins (Ccl,, acetaminophen, ethanol) and protect MAT from oxidant- induced inactivation, with the concomitant increase in the

production of AdoMet. In the liver of rats chronically fed ethanol, AdoMet has also been shown to improve the mitochondrial uptake of GSH (Garcia-Ruiz et al., 1995). AdoMet

is also capable of antagonizing the depletion of hepatic folate observed during liver injury, thus restoring normal homocysteine levels and purine metabolism (Varela-Moreiras et

al., 1995). Finally, exogenous AdoMet might spare cellular ATP, otherwise used in its synthesis, as well as NAD, and thus

prevent the depletion of these molecules and mitochondrial

de-energization induced by cell injury. The decrease in hepatic GSH levels induced by a variety

of hepatotoxins has been shown to be prevented by the ad-

ministration of AdoMet (Mato et al., 1994). Moreover, as mentioned above, the protective effect of AdoMet in a mouse model of acetaminophen-induced intoxication was blocked by inhibition of GSH synthesis with BSO (Bray et al., 1992). AdoMet administration has also been shown to

effectively increase the hepatic GSH content of patients

with alcoholic liver disease (Vendemiale et al., 1989). These results, together with the finding that N-acetylcys-

teine, a precursor of GSH synthesis, also impedes acetaminophen-induced liver injury (Corcoran and Wong, 1986;

Bray et al., 1992), strongly suggests that AdoMet exerts its function, at least in part, by preventing GSH depletion, working both as a GSH precursor and as an activator of the transsulfuration pathway.

In summary, the protective effect of AdoMet, therefore,

could be at various levels through the facilitation of methylation reactions, sparing ATP and NAD, preventing GSH depletion, and restoring normal folate metabolism. In addition, AdoMet could act extracellularly, perhaps through the methylation of phospholipids and/or proteins, or through interactions with purinergic receptors. The relative contribution of each pathway may vary depending on the nature of the hepatotoxic agent, experimental model, and other vari- ables. Nevertheless, liver injury and the progression of the liver disease are clearly associated with an impaired synthesis and metabolism of AdoMet. To understand the factors


that may be involved in the abnormal production of AdoMet, it is helpful to review our present knowledge of

the molecular biology of the synthesis of this molecule.

3. MOLECULAR BIOLOGY OF S-ADENOSYLMETHIONLNE SYNTHESIS 3.1. Different Forms of

Methionine Adenosyltransferase in Mammals

The existence of isozymes of MAT was first demonstrated in S. cerevisiae by Chiang and Cantoni (1997). In mamma-

lian tissues, three isozymes of MAT (MAT I, MAT II and

MAT III) have been identified. The situation encountered in mammals, therefore, is similar to the one found in Es- cherichia coli or in S. cereuisiue, where multiple and different

forms of MAT have also been identified (Markham et al., 1984; Satishchandran et al., 1990; Thomas and Surdin-Ker- jan, 1987; Thomas et al., 1988). The cloning and sequenc-

ing of the structural genes or cDNAs encoding for a large variety of MATS, including Mycoplasma genitdium, E. coli, S. cereuisim, Arubidopsis thliana, human, rat and mice liver,

and human and rat kidney, show that despite the biochemical variability of forms, MAT is an exceptionally well-con-

served enzyme through evolution. MAT I and MAT III (known also as MAT (Y and MAT l3) are a homotetramer

and a homodimer, respectively, of the same subunit (the (Y- subunit) (Cabrero and Alemany, 1988; Alvarez et al.,

1994), and are expressed exclusively in the liver (Alvarez et al., 1993). The deduced amino acid sequence of the cw-subunit corresponds to polypeptides with an M, of 43,647 and 43,697 for the human and rat liver enzymes, respectively (Horikawa et al., 1989; Horikawa and Tsukada, 1991; Alva-

rez et al., 1991, 1993). MAT I and MAT III have distinct kinetic properties for methionine and ATP. Values for

K mMet ad KWTP have been calculated by many laboratories,

and differ depending on the experimental conditions and with the method used for the purification of the enzyme

(Lombardini et al., 1973; Liau et al., 1977; Kunz et al., 1980; Sullivan and Hoffman, 1983; Hoffman, 1983; Cabrero et al., 1987; Pajares et al., 1992b). Under a given set of experimental conditions, Pajares et al. (1992b) have reported a hyperbolic kinetics for both substrates, methionine and ATP, using purified rat liver MAT I and MAT III. The K, value for methionine of MAT I and MAT III was 125 PM

and 650 @I, respectively, and the K, value for ATP of MAT I and MAT III was 250 FM and 900 PM, respectively (Pajares et al., 199213). MAT I and MAT III also differ in their response to dimethylsulfoxide (DMSO). While MAT III is activated by DMSO about 50-fold, MAT I only increases its activity slightly in response to this agent (Hoff- ,man and Sullivan, 1982; Okada et al., 1981; Suma et al., 1983). MAT II p is resent in many tissues, including brain, kidney, testis, lymphocytes, fetal liver and, in small amounts, in adult liver (Kotb and Geller, 1993). MAT II has lower K, values for methionine than MAT I and MAT III (Kotb and Geller, 1993) and is inhibited by DMSO (Okada et al., 1980).

Earlier studies revealed that marked changes in MAT activity occur during liver development. Thus, in mouse liver, Hancock (1966) described a rapid rise in MAT activity after birth from trace activities in the near-term fetus to a

peak value at age 21 days. Finkelstein (1967) also reported a significantly lower MAT activity in the suckling and weanling rat than that in the adult rat. In a comprehensive study, Chase et al. (1968) found that MAT specific activity began to increase in late fetal life, reaching a maximum 2

days after birth, and decreasing slightly by adulthood. Changes in liver MAT isoenzymes during development

have also been reported. Based on their respective kinetic properties and on immunohistochemical analysis, it has been shown that MAT I and MAT III are expressed only in adult liver, while MAT II is predominantly expressed in fe-

tal liver and faintly detected in the adult organ (Okada et al., 1980; Horikawa et al., 1993). In humans, it has also been reported that MAT II is weakly expressed in adult liver and, in contrast to the rat, MAT I and MAT III isoen-

zymes are present in low levels in fetal liver (Liau et al., 1979; Horikawa and Tsukada, 1992; De la Rosa et al., 1995). In rats, the concentration of mRNA in the liver-

specific MAT increases from the earliest fetal age studied, rises strikingly after birth, and peaks at age 10 days, main-

taining these levels during the adult life (Gil et al., 1996). Conversely, mRNA levels of the MAT II isoenzyme decrease gradually until birth and reach a minimum in adult

life (Gil et al., 1996). This switch in the predominant expression from MAT II to the liver-specific isoenzymes MAT I and MAT III in rat liver during late fetal life seems to be regulated by cyclic AMP (Gil et al., 1996). Interest-

ingly, rat hepatocytes mimic isoform switching, indicating that both mRNAs are differentially regulated in the same cell type. This finding excludes the possibility that changes

observed in the content of MAT II mRNA in total rat liver

could be due to the contribution of hematopoietic cells present in different proportions in each fetal stage. Gil et al.

(1996) also concluded that the presence of liver-specific MAT protein or mRNA are markers of the differentiated state of the hepatocyte as good as cr-fetoprotein or albumin.

In contrast to the liver MAT I and MAT III isoenzymes that represent two polymeric forms of the same subunit (Alvarez et al., 1994), MAT II has been proposed to be a tetramer of two different subunits: a catalytic o-subunit and

a regulatory P-subunit (Kotb and Kredich, 1985; Mitsui et al., 1988; De la Rosa et al., 1992a,b). The human and rat kidney MAT II o-subunits have been cloned and se- quenced (Horikawa et al., 1990; Horikawa and Tsukada, 1992). The sequence similarity between the kidney and liver o-subunits is high. Thus, human liver and kidney a-sub-

units display sequence similarities of 84%, and a comparable value is found between the rat liver and kidney a-subunits. Rat and human liver a-subunits display a sequence similarity of 95% (Alvarez et al., 1991, 1993).

Evidence for the existence of a regulatory p-subunit in MAT II has been given in various tissues, including human lymphocytes and native erythrocytes, and bovine brain

Synthesis of S-adenosylmethionine

(Kotb and Kredich, 1985; Mitsui et al., 1988; De la Rosa et al., 1992a,b). The p-subunit is unrelated to the a-subunit with respect to peptide maps and immunoreactivity (Kotb and Kredich, 1985; Kotb et al., 1990), and it has been pro-

posed that the p-subunit may have a regulatory function (Kotb and Geller, 1993). The fact that the expression of the nontissue-specific a form in E. coli yields an active MAT (De la Rosa et al., 1995) indicates that the p form is

not necessary for activity. Cloning and sequencing of the p form, as well as co-expression of the OL- and p-subunits, will

be necessary to understand the exact molecular nature and role of this regulatory p-subunit.

The sharp increase in the expression of the liver-specific MAT after birth is accompanied of a S-fold increase in liver y-cystathionase activity (Hirai et al., 1994). This might re-

flect a higher demand of cysteine for GSH synthesis after

birth, and could explain why liver AdoMet content is higher in late gestation and after birth than in immediate

postnatal periods (Gil et al., 1996). Studies carried out in humans (Cabrero et al., 1988) and rats (Corrales et al.,

1992) indicate that marked alterations in hepatic MAT activity do not necessarily result in significant changes in

AdoMet levels, a result that was interpreted as indicative that the rate of utilization of this metabolite is accommo- dated to the rate of synthesis.

The gene for MAT has been identified recently in the genome of M. genitalium, a bacterial parasite of the human

genital and respiratory tract (Fraser et al., 1995). M. geni- t&m is thought to contain one of the smallest genomes for

a self-replicating organism (580 kb) and represents an important system for exploring the minimal set of functional genes necessary for independent life. The fact that MAT is

one of the 482 genes necessary for minimal independent

life further emphasizes the relevance of this enzyme to maintaining cell structure and function.

3.2. Structure 4 the Liver-Specific Methionine Adenosyltransferase

As mentioned in Section 3.1, the liver-specific MAT is present

in two oligomeric states, a homotetramer and a homodimer, which on gel filtration chromatography show apparent mo-

lecular masses of 210 and 110 kDa, respectively, and are

composed of a 43 kDa subunit (Cabrero et al., 1987; Hoffman, 1983; Horikawa et al., 1989; Alvarez et al., 1991). Except in the liver, in all other tissues and organisms studied, MAT is present as a tetramer. The mechanisms that regulate the ra-

tio between the dimer and the tetramer in the liver, and the pathway to their synthesis, are not known. It has been shown that mutation of some cysteine residues of the central domain of the enzyme subunit (Takusagawa et al., 1996) shifts the distribution of the oligomeric forms in the rat liver enzyme (Mingorance et al., 1996). Substitution of Cys-69 modified the structure of the native enzyme, yield- % primarily dimers, and substitution of Cys-35, Cys-61 or Cys-105 shifted the dimer/tetramer equilibrium, yielding mainly dimers (Mingorance et al., 1996). No monomeric

273

forms of the enzyme were detected in any case. In vitro,

both forms of the rat liver enzyme seem to be very stable,

and only upon treatment with high concentrations of LiBr has conversion of the tetramer into catalytically active

dimers been observed (Cabrero et al., 1988). In wiwo, changes in the relative amounts of both isoforms have been observed during development (Okada et al., 1980), and also in certain experimental models, such as rats fed with me-

thionine- or ethionine-rich diets (Matsumoto et al., 1984; Tsukada et al., 1980), in N-2-fluorenylacetamide-induced

carcinogenesis (Tsukada et al., 1980), and in some pathological conditions in humans (Cabrero et al., 1988). Disrup-

tion of rat liver MAT II into catalytically active monomers has been obtained by phosphorylation with protein kinase C (Pajares et al., 1994). A model for the regulation of liver MAT, based on the results obtained in vitro with the puri-

fied MAT I and MAT III forms, was proposed by our laboratory (Pajares et al., 1992a).

Incubation of hepatic MAT (MAT I and MAT III) with

sulfhydryl group reagents markedly reduces the enzyme activity. p-Chloromercuribenzoate (Lombardini et al., 1973),

p-chloromercuriphenyl sulfonate (Liau et al., 1977), fumarylacetoacetate (Berger et al., 1983; Berger, 1985), N-ethyl- maleimide (Corrales et al., 1990; Pajares et al., 1991) and

GSSG (Pajares et al., 1992b), all have been shown to inhibit MAT I and MAT III. Hepatic MAT inhibition by fumarylacetoacetate or GSSG was prevented by the addition of

dithiothreitol and GSH, respectively (Pajares et al., 199213; Berger et al., 1983). MAT II differs from MAT I and MAT III by its resistance to inhibition by sulphydryl reagents.

Thus, whereas MAT I and MAT III are completely inhibited by pchloromercuribenzoate or fumarylacetoacetate,

MAT II is resistant to these agents (Okada et al., 1980; Berger et al., 1983; Berger, 1985).

The importance of sulfhydryl groups to maintain MAT activity in viva has been discussed in Section 2.2. Cys-121

was identified as the site of molecular interaction between NO (and probably other sulphydryl reactive reagents, such as hydroxyl radicals or GSSG) and rat liver MAT, which is responsible for the inhibition of the enzyme. To reach this conclusion, each of the 10 cysteine residues of the a-subunit

of the liver-specific MAT were individually changed to

serines by site-directed mutagenesis, and the effect of NO on the various recombinant enzymes tested. Replacement

of Cys- 12 1 by serine produced an enzyme that was resistant to NO inhibition. None of the other nine mutants were resistant to NO inhibition (Mingorance et al., 1996; Avila et

al., 1996). Cys-121 is localized at a “flexible loop” over the active site cleft of MAT (Takusagawa et al., 1996). Cys-121 is typical of rat, mouse and human liver-specific enzymes, but is not present in any of the other known sequences of MAT, including rat and human kidney, E. cob, yeast or Drosophila (Mingorance et al., 1996). This agrees with the observation, mentioned above, that MAT II is resistant to sulfhydryl group reagents.

The active site of rat liver MAT I and MAT III forms has been studied using 8-azido ATP, a photolabile analogue of


the substrate (Deigner et al., 1995). This experiment al-

lowed us to identify a peptide, amino acids 267-286 of the rat liver enzyme, that presumably binds ATP close to posi- tion 8 of the adenosine ring, and that might be the active site of MAT. This peptide has been shown to be extremely

well conserved in all the sequences of MAT available at the moment. At the same time, this peptide contains what is

known as the “P-loop”, a consensus sequence present in several families of ATP binding proteins for phosphoryl group binding (Deigner et al., 1995).

3.3. Genetic Deficiency in

Heputic Methionine Adenosyltransferase

Genetic deficiency in hepatic MAT has been suggested in

seven patients by showing diminished MAT activity in liver biopsies and persistent hypermethioninemia as com-

pared with control individuals (Gaul1 and Tallan, 1974; Finkelstein et al., 197513; Gaul1 et al., 1981; Gahl et al.,

1987). Several mutations have been identified in the coding region of the MAT gene of three of these patients with enzymatically confirmed diagnosis of MAT deficiency (Ubagai et al., 1995). In one patient, a mutation that converts Ile-

322 to Met was found; in a second patient, a compound heterozygote with different mutations, Ala-55, was substi-

tuted by an Asp and Pro-357 by Leu; and in the third patient, also a compound heterozygote, a mutation converting

Leu-305 to a Pro, and a second mutation that converts Ile- 322 to a Met were found. Ala-55, Ile-322 and Pro-357 have

been virtually invariant during evolution, and in the case of Leu-305, only conservative changes to Ile (in bacteria) or

Val (in plants) have arisen, suggesting that these residues are important to maintain MAT structure and/or activity. To confirm this point, four mutants have been constructed, each

bearing one of the four mutations identified in the patients. Upon expression in COS-1 cells, all 4 mutants were found to

have lower activity (3- to 6-fold) than COS-1 cell transfec- tants bearing the wild-type MAT cDNA (Ubagai et al., 1995).

Individuals with hepatic MAT deficiency are character-

ized by isolated persistent hypermethioninemia (Ubagai et

al., 1994). The pl asma methionine concentration in he-

patic MAT-deficient patients can be up to 35-fold higher than the upper limit in normal adult individuals (about 35 PM). In some patients, an unusual breath odour occurs due

to the presence of unusually large amounts of dimethylsul- fide (Gahl et al., 1987). Interestingly, transgenic plants with suppressed MAT activity had an abnormal phenotype and a characteristic smell, a consequence of the accumulation of methionine that is converted into the volatile

methanethiol (Boerjan et al., 1994). These individuals with persistent hypermethioninemia

due to a hepatic MAT deficiency are free from homocysteinemia, tyrosinemia or serious liver disease, signs of conditions where hypermethioninemia is a secondary effect (Boerjan et al., 1994). The absence of serious liver disease in these patients indicates that liver disease cannot be the direct consequence of a deficient synthesis of AdoMet.

However, one of these patients with genetic deficiency of hepatic MAT developed abnormal neurological symptoms and neural demyelination, and both manifestations responded favourably to the administration of AdoMet (Sur-

tees et al., 1991). This indicates that isolated persistent hypermethioninemia may not always be a benign condition.

How do we reconcile the fact that liver disease is protected by AdoMet therapy with the finding that there is no serious liver disease in patients with a genetic deficiency in

MAT? As mentioned in Section 2.2, MAT deficiency in liver disease may be secondary to the generation of free radicals (NO, OH) and oxidants (GSSG), and a mechanism to spare ATP and maintain NAD content, and consequently, prevent cellular de-energization (SQnchez-Gbngora et al.,

1996). Membrane and DNA damage during oxidative stress, as well as the depletion of GSH and the generation

of toxic species in liver injury, together with the repairing role of AdoMet in these processes, might well be the reason

for the hepatoprotective effect of AdoMet therapy.

3.4. Regulation and Structure of the

Liver-Specific Methionine Adenosyltransferase Gene

The organization of the liver-specific human MAT gene re-

cently has been described, and it has been shown to contain 9 exons and 8 introns (Ubagai et al., 1995). The exons were

found to account for the full coding region in the cDNA. The gene for rat liver-specific MAT synthetase is present

as a single copy in the genome (Alvarez et al., 1993), as it also has been reported for its human (Ubagai et al., 1995)

and mouse (Sakata et al., 1993) counterparts. The major start site of transcription in the rat was determined to be 29

nucleotides downstream of the putative TATA sequence (Alvarez et al., 1993), at the same site as that for the mouse MAT gene (Sakata et al., 1993), further supporting the as- signation of this nucleotide as the cap site.

By analyzing a series of 5’send deletions, several regulatory regions involved in the tissue-specific promoter activ-

ity have been identified. Most notably, promoter elements located between - 1405 and -958 bp produced the highest

reporter activity upon transfection in H35 or HepG2 cells, but had a negative effect in CHO cells (Pajares et al., 1996). Sequence analysis of this region revealed the presence of consensus binding sites for both ubiquitous (AP-1, NF- 1) and liver-enriched (HNF- 1, HNF-3) transcription factors. Interestingly, HNF-1 and HNF-3 elements are tan-

demly arrayed and separated by only 13 nucleotides. It is well documented that the successful expression of several liver-specific genes requires a combined action of ubiquitous factors, such as NF-1, and liver-enriched factors (Sladek and Darnell, 1992; Tronche and Yaniv, 1992; Xan- thopoulos and Mirkovitch, 1993; Zaret, 1994). Therefore, it is conceivable that the putative recognition motifs found between - 1405 and -958 bp account for transcriptional efficiency of liver-specific rat MAT gene. In fact, removal of this region leads to a drastic reduction in promoter activity. Nevertheless, further studies are required to verify whether


these elements, either alone or in a combined fashion, are

mediating liver-specific rat MAT promoter activity.

In contrast with the expression pattern obtained in the hepatic-type cells, the - 1405 bp/-958 bp region appears to influence negatively the promoter function in CHO cells, since its deletion resulted in an increased level of transcrip-

tional activity (Pajares et al., 1996). This result suggests the existence in this area of a tissue-specific negative regulatory element, as it has been described for many other genes, including insulin (Nir et al., 1986), ol-fetoprotein (Muglia and Rothman-Denes, 1986), albumin (Herbst et al., 1989),

apolipoprotein CIII (R eue et al., 1988) and immunoglobu-

lin heavy chain (Imler et al., 1987). A negatively acting region also has been identified in the mouse MAT promoter.

However, in this case, the negative regulatory element is located between - 5 18 and 366 bp, and appears to be func-

tional in hepatic cells, not having been analyzed in other cell types (Sakata et al., 1993).

Another region of importance in the 5’-flanking region

of liver-specific rat MAT gene is located between -193 and -87 bp, since its deletion produced a significant decrease in promoter activity (Pajares et al., 1996). Surpris- ingly, this region contains few sequences that match the

consensus binding sites for known transcription factors. In fact, only a putative HNF-4 motif was detected, which could also account for the transcriptional efficiency of the

liver-specific rat MAT promoter. Sequences from -958 to -727 and -375 to - 193 seem to act as positive regulatory

elements in CHO cells. Consensus motifs identified in these areas correspond to binding sites for widely distrib- uted transcription factors such as AP- 1. Thus, differences in promoter activity detected in the different cell types upon

transfection with these deletion constructs may be due to the relative concentration and ratios of ubiquitous factors in particular cells, as has been found in many other genes (Diamond et al., 1990; Mietus-Snyder et al., 1992; Liu et al.,

1993). On the other hand, the region located between nucleotides -87 and +65, containing minimal promoter ele-

ments, exhibited similar relative transcriptional activity in the 3 cell lines tested (H35, HepG2, CHO). It suggests that

this fragment contains positive elements commonly active in both cell types, probably basic regulatory sequences such as the TATA box.

It is interesting to note that main differences can be found when the functional analysis of the 5’-flanking re-

gion of liver-specific rat MAT gene is compared with that reported for its mouse counterpart, despite the high se-

quence similarity in the 1113 bp overlapping regions. Basi- cally, in the mouse promoter, the 5’ &-acting elements, which produce the highest transcriptional activity, are lo-

cated between -365 and -145 bp (Sakata et al., 1993), while in the rat promoter, the region responsible for the maximal activity is comprised between -1405 and -958 bp. It is not known whether the crucial distal elements found in the rat promoter are also present in its mouse

counterpart, since in the latter, this region has not been se-

quenced or analyzed.

Previous studies in adrenalectomized rats showed that

glucocorticoids stimulate hepatic MAT activity (Pan and

Tarver, 1967; Pan et al., 1968a,b; Shou et al., 1968). Re-

cently, we have given evidence indicating that glucocorticoids have a direct effect on the expression of the liver-spe-

cific gene (Gil et al., 1997). Triamcinolone was found to modulate the expression of the endogenous liver-specific MAT gene in H35 cultured cells, as well as in cultured rat hepatocytes, by increasing the level of the corresponding

mRNA in a time- and dose-dependent manner. Glucocorti- coid-dependent expression of the liver-specific MAT gene has been confirmed by the finding that the promoter activity is also induced by triamcinolone. In addition, a region

conferring glucocorticoid inducibility has been identified

between nucleotides -727 and -527. Consistent with this

observation, two glucocorticoid responsive elements (GRE) half-sites have been identified in the liver-specific rat MAT

promoter in this area. Although these sequences do not match with the canonical GRE palindrome, it is well documented that the glucocorticoid receptor, upon binding the hormone, can efficiently interact with an imperfect palindrome (Truss and Beato, 1993). It is also worth noting that a putative GRE identified in this region overlaps with a

consensus binding site for the transcription factor NF-1. In this regard, it has been reported that the glucocorticoid re-

ceptor is able to act synergistically with NF-1 to increase transcriptional activity (Truss and Beato, 1993). It remains

to be determined whether these putative GREs are mediat-

ing the glucocorticoid responsiveness of the rat liver MAT gene, as well as the possible involvement of NF- 1.

Acknowkdgemenrs-This work was supported by grants from Fonda de In- vestigaciones Sanitarias (95/0966), D’ lreccion General de Investigation Cientifica y Tecnologica (PB 94/0087), Comunidad Autonoma de Madrtd, European Union Science Programme (SCI*-CT92-0780), Plan National de Salud (SAF 96-0108) and Europharma S.A.

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