1 Stokes Research Institute-Children's Hospital of Philadelphia; and 2 Department of Biochemistry and Biophysics, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
THE CLASSICAL SIGNALING PATHWAY for
nitric oxide (NO) is the binding and subsequent activation of soluble
guanylate cyclase. However, the number of pathophysiological pathways
mediated by NO has been expanding to include processes that are
independent of guanylate cyclase. A number of alternative reaction
pathways for NO have been proposed to account for these effects, most
notably S-nitrosylation (12). In the study by
St. Croix et al., one of this issue's articles in focus (Ref. 11, see
p. L185), the authors propose a novel mechanism for NO-based signaling
through the regulation of zinc homeostasis. Central to this proposal is the cysteine-rich protein thionein (T), which, due to the high density
of reduced cysteine residues, is an extremely avid binder of divalent
cations in general and specifically of zinc. The metal bound form of T,
metallothionein (MT), is capable of binding up to seven
divalent cations in thiolate clusters. The ability of MT to retain its
metal is critically dependent on the redox state of its cysteine
residues (9). In their paper, St. Croix et al.
(11) demonstrate that within a cell, NO exposure results in MT-based release of zinc.
Using the lipophilic compound zinquin, which increases its fluorescence
upon zinc binding, the authors are able to monitor alterations in the
intracellular concentration of free zinc upon exposure to NO. By
examining fibroblasts from MT knockout and heterozygous mice, they
demonstrate that MT is necessary for NO-mediated increases in
intracellular free zinc. Furthermore, NO effects on zinc metabolism are
restored by transfection of MT These latter observations raise some interesting questions as to the
molecular mechanism of NO-mediated release of zinc from MT. Perhaps
most critical among them is the chemical nature of the modification(s)
of the protein itself, which results in zinc release and conformational
change. The nature of the modification of MT induced by NO is critical
to understanding how the reported MT/T balance operates to control zinc
responses to NO exposure. For instance, does overexpressed T inhibit
NO-induced action by rebinding released zinc or by operating as a sink
for NO; i.e., could similar inhibition have been achieved with a
non-zinc-binding thiol (although such inhibition would clearly not be
zinc reversible)? The most obvious suggestion for the mechanism of
NO-induced zinc release is the formation of S-nitrosothiol
either in the metal binding center of the protein or in some other
cysteine residue, which induces a conformational change resulting in
zinc release. Significantly, the NO donor used in this study was
S-nitrosocysteine, implying that this may be an
S-nitrosothiol/nitrosonium-ion mediated pathway. It would be
interesting in future studies to investigate how other redox congeners
of NO affected this system. Previous experiments have shown that MT can
be induced to release zinc upon cysteine oxidation to intramolecular
disulfides (9). NO is capable of inducing cysteine
oxidation as well as nitrosylation; therefore, it remains to be seen
whether NO-mediated release occurs via different molecular mechanisms.
This is significant because it may reveal convergent but alternative
mechanisms for oxidative and nitrosative stress within intracellular signaling.
Although zinc has been shown to operate as a signaling molecule
(1) and as a mediator of cell death (6),
these functions have been mediated via flux of extracellular ions. The
present study brings to bear a whole new dynamic of the regulation of intracellular zinc homoeostasis. A major key to understanding the
relevancy of this control mechanism is to identify the cellular targets
of MT-released zinc. A number of proteins incorporate zinc as a
structural component ranging from enzymes (superoxide dismutase) to
transcription factors (TFIIIA) to steroid-thyroid hormone receptors
(BRCA1) (3). Interestingly, even NO synthase has been
recently shown to contain structurally important zinc (4,
10). Entry of extracellular zinc into GH3 cells has been shown
to produce transcriptional activation (2), and zinc
removal from inhibitory sites has been shown to activate a number of
enzymes, including caspase 3 and glyceraldehyde-3-phosphate
dehydrogenase (8). Recently, it was proposed that zinc
release from MT inhibits mitochondrial respiration (13),
providing a mechanism for redox modulation of mitochondrial
bioenergetics. In this way the MT/T balance, which would reflect both
zinc concentrations and redox status of the protein, would operate both
as a controller and as a sensor of cellular redox status.
Zinc, as opposed to other biological cations such as iron and copper,
is a redox-inactive ion. Therefore, the data presented in this paper
suggest a novel and interesting paradigm in which the versatile
biological signal molecule NO, which can exist in multiple redox
states, regulates the intracellular homeostasis of an entirely
redox-insensitive molecule, zinc. This represents a novel mechanism of
energy transduction, i.e., the redox chemical energy of NO is
transduced to the structural chemical energy of zinc. In this paradigm,
MT represents a key transducer of redox energy and provides a novel
mechanism for redox regulation in cellular homeostasis.
There is a strong similarity between NO and zinc function within
pathophysiology, namely that they can both be cytoprotective and
cytotoxic. Zinc has been implicated in neuronal death (5, 6) and yet it is necessary for normal cellular function and has
been shown to inhibit proapoptotic proteins, such as caspase 3. Interestingly, NO has also been implicated in cell death following ischemia, is necessary in a number of cellular functions, and has been shown to inhibit caspase 3 (7). Of critical
importance to determine the effects of NO exposure is where it is made,
how much is made, and how fast it reacts with molecular targets. It may
be that the same principles can be applied to the MT/T-mediated regulation of zinc and the role that NO plays in controlling the zinc
homeostatic system.
ARTICLE
TOP
ARTICLE
REFERENCES
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fibroblasts with MT expressing
adenoviral vector. Having established that MT is essential to this
NO-mediated effect, St. Croix et al. further demonstrate that zinc
levels are critically dependent on the balance between MT and T. Overexpression of MT in sheep pulmonary artery endothelial cells
results in an inhibition of NO-mediated zinc release, which can be
restored by growing the cells in media containing high concentrations
of zinc. The supposition here is that overexpression of MT in the
absence of excess zinc results in a large proportion of MT being in its
apo-form T. Therefore, when there is a large intracellular
concentration of T, relative to MT, there is no NO-mediated release of
zinc. Using fluorescent chimeras of MT, St. Croix et al. showed that MT
overexpression inhibited NO-induced conformational changes in MT
associated with zinc release and that this inhibition could be
alleviated by growth in zinc-rich medium.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Ischiropoulos, Dept. of Biochemistry and Biophysics, The Univ. of Pennsylvania School of Medicine, Philadelphia, PA 19104 (E-mail: ischirop{at}mail.med.upenn.edu).
10.1152/ajplung.00424.2001
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REFERENCES |
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1.
Assaf, SY,
and
Chung SH.
Release of endogenous Zn2+ from brain tissue during activity.
Nature
308:
734-736,
1984[ISI][Medline].
2.
Atar, D,
Back PH,
Appel MM,
Gao WD,
and
Marban E.
Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels.
J Biol Chem
270:
2473-2477,
1995
3.
Berg, JM,
and
Shi Y.
The galvanization of biology: a growing appreciation for the roles of zinc.
Science
271:
1081-1085,
1996[Abstract].
4.
Ghosh, DK,
Crane BR,
Ghosh S,
Wolan D,
Gachhui R,
Crooks C,
Presta A,
Tainer JA,
Getzoff ED,
and
Stuehr DJ.
Inducible nitric oxide synthase: role of the N-terminal -hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction.
EMBO J
18:
6260-6270,
1999
5.
Jiang D, Sullivan PG, Sensi SL, Steward O, and Weiss JH.
Zn2+ induces permeability transition pore opening and
release of pro-apoptotic peptides from neuronal mitochondria.
J Biol Chem. In press.
6.
Koh, JY,
Suh SW,
Gwag BJ,
He YY,
Hsu CY,
and
Choi DW.
The role of zinc in selective neuronal death after transient global cerebral ischemia.
Science
272:
1013-1016,
1996[Abstract].
7.
Mannick, JB,
Hausladen A,
Liu L,
Hess DT,
Zeng M,
Miao QX,
Kane LS,
Gow AJ,
and
Stamler JS.
Fas-induced caspase denitrosylation.
Science
284:
651-654,
1999
8.
Maret, W,
Jacob C,
Vallee BL,
and
Fischer EH.
Inhibitory sites in enzymes: zinc removal and reactivation by thionein.
Proc Natl Acad Sci USA
96:
1936-1940,
1999
9.
Maret, W,
and
Vallee BL.
Thioloate ligands in metallothionein confer redox activity on zinc clusters.
Proc Natl Acad Sci USA
95:
3478-3482,
1998
10.
Raman, CS,
Li H,
Martasek P,
Kral V,
Masters BS,
and
Poulos TL.
Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center.
Cell
95:
939-950,
1998[ISI][Medline].
11.
St. Croix, CM,
Wasserloos KJ,
Dineley KE,
Reynolds IJ,
Levitan ES,
and
Pitt BR.
Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein.
Am J Physiol Lung Cell Mol Physiol
282:
L185-L192,
2002
12.
Stamler, JS,
Lamas S,
and
Fang FC.
Nitrosylation: the prototypic redox-based signaling mechanism.
Cell
106:
675-683,
2001[ISI][Medline].
13.
Ye, B,
Maret W,
and
Vallee BL.
Zinc metallothionein imported into liver mitochondria modulates respiration.
Proc Natl Acad Sci USA
98:
2317-2322,
2001