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Department of Microbiology & Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ 07103
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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KEY WORDS: • homocysteine thiolactone • homocysteine • protein homocysteinylation • HDL-associated thiolactonase • calcium • atherosclerosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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, Riegel and Du Vigneaud 1935
), the first indication of its biological significance came
almost 50 years later with the discovery of enzymatic conversion of Hcy
to Hcy thiolactone in error-editing reactions of some
aminoacyl-tRNA synthetases (AARS) in vitro (Jakubowski and Fersht 1981
) and in vivo (Jakubowski 1990
). Hcy
thiolactone reacts easily with proteins. Protein damage caused by
homocysteinylation may underlie the involvement of Hcy in human
pathologies such as vascular disease (Jakubowski 1997
). |
Mechanisms of homocysteine thiolactone synthesis |
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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, 1991
, 1995
and 1997
, Jakubowski and Goldman 1993
). Because Hcy thiolactone forms at the active
site of MetRS, the synthesis of thiolactone increases with an increase
in the Hcy/Met ratio. Two other synthetases, LeuRS and IleRS, can also
convert Hcy to the thiolactone under some conditions in bacteria
(Jakubowski 1995
). The mechanism of Hcy thiolactone
synthesis involves a two-step reaction driven by the hydrolysis of
ATP (Jakubowski and Fersht 1981
). In the first step (Eq. 1)
, a carboxyl group of Hcy is activated by ATP, forming a
MetRS-bound homocysteinyl adenylate.
 | (1) |
In the second step (Eq. 2)
, the side chain thiolate of Hcy
displaces the AMP group from the activated carboxyl group of Hcy,
forming Hcy thiolactone as a product. The energy of the anhydride bond
of Hcy~AMP is conserved in the intramolecular thioester bond of Hcy
thiolactone.
|
| (2) |
Hcy thiolactone is synthesized by human
endothelial cells (Table 1
), fibroblasts, breast cancer cells (Table 2
), HeLa cells, as well as by normal BALB/c 3T3 and transformed RAG mouse
cells (Jakubowski and Goldman 1993
). MetRS mutants of
Chinese hamster ovary cells, defective in the Met binding site of the
enzyme, are also defective in Hcy thiolactone synthesis
(Jakubowski and Goldman 1993
). Methionine inhibits
synthesis of Hcy thiolactone in rodent and human cells (Jakubowski,
unpublished data). Because of its mostly neutral character at
physiologic pH (pK = 7.1; Anderson and Packer 1974
), Hcy thiolactone diffuses through cell membranes and
accumulates in the culture medium (Fig. 1
).
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Elevation of Hcy levels leads to enhanced synthesis of the thiolactone |
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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. However, if Hcy levels are increased because
of a reduction in its transmethylation and/or transsulfuration, the
synthesis of Hcy thiolactone is enhanced. For example, in the absence
of folate, human fibroblasts, breast cancer cells and endothelial cells
accumulate Hcy and synthesize large amounts of the thiolactone (Table 2)
. Thiolactone levels can reach as much as 60% of the metabolized Hcy
in extreme cases of intercellular Hcy elevation, such as observed in
human endothelial cells maintained on Hcy in Met-free media
deprived of vitamin B-12 and folate (Table 2)
. |
Metabolism of Hcy thiolactone in human cell cultures and serum |
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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, Hcy thiolactone undergoes the following two
major reactions in human cells and serum in vitro: 1)
protein homocysteinylation at lysine residues (Jakubowski 1997
, 1999a
and 1999b
); and 2) enzymatic hydrolysis to Hcy
by calcium-dependent Hcy thiolactonase, a component of HDL
(Jakubowski 1999a
). Preliminary experiments suggest that
protein homocysteinylation may occur in humans. For example, small
amounts of Hcy are present in acid hydrolyzates of dithiothreitol
(DTT)-treated human serum proteins from normal subjects; more Hcy is
recovered from serum proteins from homocysteinuric subjects
(Table 3
). Enzymatic hydrolysis of thiolactone to Hcy also occurs in vivo; this
was shown by means of dietary supplementation or injection of Hcy
thiolactone as a source of Hcy in laboratory animals. Although Hcy
thiolactone is eliminated rapidly from blood and cells (the
half-life of exogenous Hcy thiolactone is 1 h or less;
Donahue et al. 1974
, Dudman and Wilken 1981
, Dudman et al. 1991
, Jakubowski 1997
), small amounts of Hcy are present in human serum proteins
(Table 3)
. Because of its reactivity, thiolactone is unlikely to be
detected in vivo (Donahue et al. 1974
, Dudman et al. 1991
, Mudd et al. 1989
). In human serum,
about half of the exogenous thiolactone incorporated into protein is
released as free Hcy after reduction with DTT. The other half
represents Hcy attached via an amide bond between its carboxyl group
and the amino group of a protein lysine residue (Fig. 2
).
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Protein homocysteinylation |
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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). In recent experiments with
[35S]Met-labeled human umbilical vein artery
endothelial cells (HUVEC), in which methionine synthase was severely
inhibited by deprivation of vitamin B-12 and folate, Hcy incorporation
into protein represented up to 36% of Hcy metabolized to Met (Table 2)
. When HUVEC were labeled with [35S]Hcy,
incorporation of Hcy into protein represented up to 65% of Hcy
metabolized to Met (Table 2)
. Hcy was present in both cellular and
extracellular proteins (Fig. 3
, Table 1
). Data suggest that Hcy incorporation into protein is
post-translational, reflecting facile homocysteinylation of protein
lysine residues by Hcy thiolactone (Fig. 2)
. Indeed,
phenylthiohydantoin (PTH)-(S-carboxymethyl)homocysteine was
recovered from tissue culture proteins subjected to carboxymethylation
and Edman degradation (Jakubowski, unpublished data). Translational
incorporation of Hcy into protein is unlikely because AARS do not
aminoacylate tRNA with Hcy (Jakubowski 1999c
and 1999d
).
|
). In human serum incubated with the thiolactone, protein
homocysteinylation is a major reaction, which could be observed with as
little as 10 nmol/L thiolactone. Individual proteins are
homocysteinylated at rates proportional to their lysine contents
(Jakubowski 1999b
). Examples of time courses of protein
homocysteinylation are shown in Figure 4
. Homocysteinylation results in protein damage, manifested as loss of
function. For example, methionyl-tRNA synthetase (Fig. 5
) and trypsin (Jakubowski 1999b
) are inactivated by
homocysteinylation. Lysine oxidase, an important enzyme responsible for
post-translational modification essential for the biogenesis of
connective tissue matrices, is also inactivated irreversibly by Hcy
thiolactone (Liu et al. 1997
).
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HDL-associated Hcy thiolactonase in human serum |
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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). The thiolactonase requires calcium for activity and
stability, and is associated with the HDL fraction of serum
lipoproteins (Table 5
). The enzyme is inhibited noncompetitively by
isoleucine (Ki = 2 mmol/L) and
penicillamine (Ki = 0.2 mmol/L). In
the absence of detergents, the thiolactonase copurifies with
apolipoprotein AI. Purification to homogeneity can be achieved only in
the presence of nonionic detergents, such as Igepal C-630. Purified Hcy
thiolactonase migrates as a protein of molecular weight 45 kDa on
denaturing polyacrylamide gels. The enzyme exhibits a remarkable degree
of substrate specificity toward Hcy thiolactone. Although homoserine
lactone is also a substrate, it is not known to occur in human cells.
Other related thioesters, such as N-acetyl-Hcy thiolactone
and acetyl-S-coenzyme A, or esters, such as
O-acetyl-serine, methyl or ethyl esters of methionine,
phenylalanine, alanine, ß-alanine and cysteine, are not substrates.
However, the thiolactonase also hydrolyzes nonnatural substrates, such
as phenyl acetate, p-nitrophenol acetate and the
organophosphate paraoxon (Jakubowski 1999a
). Extensive
research has focused on serum arylesterase/paraoxonase, an
organophosphate-detoxifying enzyme whose natural substrate and
function remain unknown (Mackness et al. 1998
). The
thiolactonase and arylesterase/paraoxonase activities comigrate at all
steps of purification, suggesting that the three activities represent
that same enzyme. These data suggest that thiolactonase/paraoxonase
protects proteins against homocysteinylation by detoxifying Hcy
thiolactone (Jakubowski 1999a
). The finding that Hcy
thiolactonase is tightly associated with HDL also suggests a link
between metabolism of Hcy and lipoproteins. This link may explain the
recent unexpected finding that paraoxonase prevents atherosclerosis in
a mouse model (Shih et al. 1998
).
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Summary: role of homocysteine thiolactone in human disease |
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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).
Available data suggest that Hcy can be harmful to human cells because
of its metabolic conversion to Hcy thiolactone, a reactive thioester.
This conversion occurs in all human cell types, including endothelial
cells. When methionine synthase activity is inhibited by folate or
vitamin B-12 deprivation, almost all Hcy is converted to thiolactone.
Subsequent inadvertent homocysteinylation of cellular and extracellular
proteins by Hcy thiolactone might lead to impaired function. The
metabolic conversion of Hcy to Hcy thiolactone, the reactivity of the
thiolactone toward proteins and resulting protein damage might explain
some pathologic consequences of elevated Hcy levels, including
atherosclerosis. The tight association of Hcy thiolactonase with HDL in
serum could contribute to the protective role of HDL in the human
vascular system.
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FOOTNOTES |
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2 Supported by a grant from the National Science Foundation (MCB-9724929).
3 Abbreviations used: AARS, aminoacyl-tRNA synthetase; DTT, dithiothreitol; Hcy, homocysteine; HUVEC, human umbilical vein artery endothelial cells; MetRS, methionyl-tRNA synthetase; PTH, phenylthiohydantoin.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMechanisms of homocysteine...Elevation of Hcy levels...Metabolism of Hcy thiolactone...Protein homocysteinylationHDL-associated Hcy thiolactonase...Summary: role of homocysteine...REFERENCES |
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1. Anderson R. F., Packer J. E. The radiolysis of aqueous solutions of homocysteine thiolactone. Int. J. Radiat. Phys. Chem. 1974;6:33-46
2.
Baernstein H. D. A modification of the method for determining methionine in proteins. J. Biol. Chem. 1934;106:451-456
3. Donahue S., Sturman J. A., Gaul G. Arteriosclerosis due to homocysteinemia: failure to reproduce the model in weaning rabbits. Am. J. Pathol. 1974;77:167-174[Medline]
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Dudman N.P.B., Hicks C., Lynch J. F., Wilcken D.E.L., Wang J. Homocysteine thiolactone disposal by human arterial endothelial cells and serum in vitro. Arterioscler. Thromb. 1991;11:663-670
5. Dudman N.P.B., Wilken D.E.L. Homocysteine thiolactone in experimental homocysteinemia. Biochem. Med. 1981;27:244-253
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Jacobsen D. W. Homocysteine and vitamins in cardiovascular disease. Clin. Chem. 1998;44:1833-1843
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Jakubowski H. Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 1990;87:4504-4508
8. Jakubowski H. Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in the yeast Saccharomyces cerevisiae. EMBO J 1991;10:593-598[Medline]
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Jakubowski H. Metabolism of homocysteine thiolactone in human cell cultures: possible mechanism for pathological consequences of elevated homocysteine levels. J. Biol. Chem. 1997;272:1935-1942
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Jakubowski H. Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J 1999b;13:2277-2283
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Liu G., Nellaiappan K., Kagan H. M. Irreversible inhibition of lysyl oxidase by homocysteine thiolactone and its selenium and oxygen analogues. J. Biol. Chem. 1997;272:32370-32377
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) and presence of indicated substrates: tRNA (•), Met (
), or
ATP (
). Control incubation in the absence of Hcy thiolactone is also
shown (
). (B) Relationship between the degree of homocysteinylation
(Hcy/MetRS, mol/mol) and residual enzymatic activity.