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Cell Signalling Biology Michael J. Berridge Module 2 Cell Signalling Pathways 2 70

proteins, but is first of all converted into reactive nitro- active site. This oxidized Trx is then reduced by NADPH
gen species (RNS) that are then responsible for carrying through the activity of the seleno-flavoprotein TrxR.
out the nitrosylation reaction. These nitrosylation reac- The other mechanism depends on S-nitrosoglutathione
tions are reversed by denitrosylation reactions.Manyof reductase (GSNOR), which is also known as GSH-
the functions of NO are carried out by this nitrosylation- dependent formaldehyde dehydrogenase and Class III al-
sensitive signalling pathway and there is growing evidence cohol dehydrogenase (ADH3). The GSNOR metabolizes
for nitrosylation dysfunction in disease. the GSNO, which is one of the reactive nitrogen spe-
cies (RNS), which is produced through two mechanisms.
Reactive nitrogen species (RNS) It is formed when NO interacts with GSH and is also
The reactive nitrogen species (RNS) that carry out the released following the denitrosylation of target proteins
nitrosylation reaction are formed when NO interacts (Module 2: Figure NO and cGMP signalling). The GS-
−• ), NOR uses NADH as an electron donor to irreversibly
with various acceptors such as superoxide radical (O 2
cysteine (Cys), glutathione (GSH) or transition metal ions convert GSNO into GSNHOH. In the absence of GS-
(M n + , e.g. Fe 3 + or Cu 2 + )(Module 2: Figure NO and NOR, the level of GSNO increases as does the amount
cGMP signalling): of S-nitrosylated proteins, which suggest that these SNO
proteins might be in equilibrium with GSNO and this
-
− → ONOO (peroxynitrite)
1. NO + O 2
might account for the fact that certain cellular proteins
2. NO + M n + → Mn n + -NO
seem to by constitutively nitrosylated.
3. NO + GSH → GS-NO
4. NO + Cys → Cys-NO
Redox signalling
−• forms the Cells have evolved a sophisticated mechanism of intracel-
The interaction between NO and O 2
strong oxidant peroxynitrite (ONOO ), which is very lular signalling based on localized changes in the oxidation
−
much more reactive than the two parent molecules. It can state of specific proteins. The internal environment of cells
react with electron-rich groups such as protein sulfhy- is normally highly reduced. Certain forms of stress are as-
dryls as part of the nitrosylation reaction to form the S- sociated with an increase in the oxidative state, and this
nitrosothiols (SNOs). Although peroxynitrite has a brief can induce apoptosis. It is also well known that certain
half-life (approx. 15 ms), it exists for long enough to dif- phagocytic cells, such as neutrophils, can rapidly generate
fuse through a cell and perhaps also to neighbouring cells superoxide radical (O 2 −• ) and hydrogen peroxide (H 2 O 2 )
enabling it to function as an intra- and inter-cellular mes- that are used to kill other cells during inflammatory re-
senger. sponses. In addition to these pathological effects, there is
Low molecular mass thiols, such as glutathione (GSH), increasing evidence that the redox system has been adapted
can also be nitrosylated by NO to form GS-NO. to perform a variety of signalling functions and can mod-
ulate the activity of other signalling pathways. As such,
Nitrosylation reaction they can control many cellular processes, including cell
The S-nitrosylation reaction depends upon the transfer of proliferation, apoptosis and cellular senescence.Someof
NO from one of the reactive nitrogen species (RNS) to these effects are exerted through a two-way interaction
a peptidyl cysteine thiol group of target proteins (R-SH) with Ca 2 + signalling. For example, redox signalling can
(Module 2: Figure NO and cGMP signalling): help to promote the tyrosine phosphorylation events that
generate many signalling cascades and it can modulate the
-
1. R-SH + ONOO → R-SNO
activity of the ryanodine receptors (RYRs) and inositol
2. R-SH + M n + -NO → R-SNO
1,4,5-trisphosphate receptors (InsP 3 Rs) that release Ca 2 + .
3. R-SH + GS-NO → R-SNO
Conversely, Ca 2 + can stimulate redox signalling, particu-
4. R-SH + Cys-NO → R-SNO
larly within the mitochondrion, indicating that there are
Specificity is determined by the fact that the target pro- dynamic interactions operating between these signalling
teins (R-SH) have hyperreactive cysteine groups where the pathways.
thiol moiety exists as a thiolate anion due to the presence There are two main types of redox signalling. The first
of positively charged amino acids in the immediate vicinity type is reactive oxygen species (ROS) signalling,which
of the protein chain. depends on the formation of ROS. The second type is
reactive nitrogen species (RNS) signalling, which is carried
Denitrosylation reactions out by RNS and is linked to the nitric oxide (NO)/cyclic
Denitrosylation is carried out by two main denitrosylases GMP signalling pathway.
(Module 2: Figure NO and cGMP signalling). One mech-
anism depends on the oxidoreductase thioredoxin (Trx) Reactive oxygen species (ROS) signalling
operating in concert with Trx reductase (TrxR).Inits re- ROS signalling has all the hallmarks of a classical signalling
duced state, the two cysteine residues in the highly con- mechanism. The second messengers are the reactive oxy-
served -Cys-Gly-Pro-Cys- active site are reduced and the gen species (ROS) formed in response to many agonists.
hydrogens are used to release the NO as HNO and to Reactive oxygen species (ROS) formation depends on the
convert the SNO group on the target protein into an SH stimulation of a NADPH oxidase that removes an electron
group. During this denitrosylation reaction, a disulphide from NADPH and adds it to oxygen to create superoxide
bond is formed between the vicinal cysteine residues at the radical (O 2 −• ), which is one of the ROS found in cells.

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