Download The Effect of Cysteine Persulfide on the Redox Regulation of the

The Effect of Cysteine Persulfide on the Redox Regulation of the Enzyme CD148
1
Henriquez ,
2
Lin ,
Stephanie
Joseph
Jon Fukuto
McNair Scholar1, Faculty Mentor2
Persulfides and How They are Biosynthesized
Methods
-CSE
H-S-H
pKa= 6.8
(a)
(b)
H-S - + H +
pKa= 14.1
Supernatant
37°C
0.5 hour
70
60
Invert
Occasionally
S 2- + H + (c)
Thiols (RSH), carbon-bonded sulfhydryl groups, play important roles physiologically
as nucleophiles, reductants, and metal ligands. They are so central to cellular function that
the amino acid cysteine, which contains a free thiol, is used both for protein structure and
enzymatic catalysis.
The emerging field of study of a related group, the persulfide (RSSH) is of great
interest. The chemical properties of persulfides theoretically present unique chemical
interactions. In the unprotonated state, electron-electron repulsion between the two sulfurs
increase the nucleophilicity of a persulfide, which is known as the α-effect. Theoretically, this
gives an ability to serve as a better reducing agent when compared to a thiol. This is further
evidenced by persulfides typically having 1-2 pKa units lower than the corresponding thiol,
allowing for higher concentrations of anionic species at physiological pH. Similarly, in the
protonated state, electron-electron repulsion weakens the S-S bond making it an excellent
electrophile.
50
FDP
DTT
30
Pyruvate
No reducing agents
10
Measure fluorescence at
485/528 nm for 20 min at 37°C
•CSE, which has been demonstrated to yield a significantly higher amount of cysteine
persulfide then CBS, is immobilized onto a glutathione-agarose resin through a glutathione
S-transferase (GST) tag. This is done so that the enzyme can be easily separated from the
rest of the mixture through centrifugation. CSE is still active while conjugated to the resin.
•CSE and its substrate cystine (Cys-S-S-Cys) are incubated with oxidized CD148. This is
done so that the produced cysteine persulfide can readily interact with CD148. The tube
was inverted every 5 minutes to resuspend resin, since it settles over time.
•Controls (DTT, resin, cystine,
cysteine and
..
.. vehicle) were incubated and inverted as well.
(a)
•FDP is a well characterized
for protein tyrosine phosphatases,
such as CD148.
R-S.. - substrateR-S-H
..
When dephosphorylated,
an
excitation
..at ..485nm results in an emission at 528nm, which
.. .. (b)
can be captured on aR-S-S
fluorimeter. R-S-S-H
.. ..
..
.. ..
pKa= 6.8
H-S-H
H-S - + H +
pKa= 14.1
S 2- + H + (c)
..
Cysteine persulfide (Cys-S-SH) is endogenously produced in cells from cystine (CysS-S-Cys) through the enzyme cystathionine γ-lyase (CSE) and, to a lesser extent,
cystathionine β-synthase (CBS). Both enzymes require the prosthetic group pyridoxal
phosphate (PLP) to perform this reaction, as shown in the mechanism above. Since Cys-SSH eventually degrades into H2S, and this is the only known way to biosynthesize H2S, it
has been recently proposed that the immense body of research attributed to H2S is actually
a marker for persulfide activity. The reported physiological effects of H2S are widespread,
including important body systems and tissues such as the cardiovascular, brain, and
immune systems.
40
20
..
H2S
RFU (X1000)
..
-
CD148
0
0
5
10
15
Time (min)
•Concentrations used: 1mM DTT, 1mM Pyruvate, 30uM FDP
•Pyruvate is the other product produced by CSE
•This result makes sense, as pyruvate should not affect the redox state of CD148
•Since pyruvate does not affect activity, cysteine persulfide or any derived species
must be responsible for reducing CD148
``````````````````````````````````
``````````````````````````````````
No Recovery of CD148 Activity With NaSH
``````````````````````````````````
`````
10
9
8
7
Persulfides can be made synthetically by reacting H2S, likely the anionic HS- at
physiological pH (C), with a disulfide as shown in the scheme above. However this method
is not as controlled as the enzymatic one, so it may yield various side reactions.
RFU (X1000)
..
R-S-H
.... ..
R-S-S-H
.. ..
..
..
R-S.. .. ..
R-S-S
.. ..
Pyruvate Has No Effect on CD148 Activity
Cys-S-S-Cys
6
5
DTT
4
NaSH
3
No reducing agents
2
1
0
The Role of CD148 In a Cell
B
A
Recovery of CD148 Activity With Enzymatically Derived Cysteine
Persulfide
0
5
10
15
20
Time (min)
60
B
50
R.F.U. (X 1000)
top view
Jared Snider, Ph.D. © 2013 Thermo Fisher Scientific Inc.
2
CD148 is a protein tyrosine phosphatase (PTP). This type of enzyme is responsible
for removing a phosphate group from certain cellular targets. As shown in A, removal of a
phosphate group can either turn the target on or off. In this way, phosphatases are
important for cellular regulation. Failure of this regulation may result in either a
hyperactivated cell, or an inactive cell depending on the function of the target. In T
lymphocytes, a type of immune cell, CD148 is one of the enzymes upregulated upon
stimulation of the cell in order to propagate the signal.
The active site of a general PTP is shown in B. As shown, they contain an active
cysteine residue in order to hydrolyze phosphate groups off of tyrosine. CD148 must
contain a reduced thiol to maintain their enzymatic activity, making it particularly vulnerable
to inactivation through oxidation. In this way, CD148 can be regulated through reduction
and oxidation.
40
CSE-Resin + Cystine
Resin + Cystine
30
Cystine
Cysteine
20
DTT
2
10
•Concentrations used: 100uM DTT, 100uM NaSH, 30uM FDP. All wells are buffered with
50mM HEPES pH7.2.
•The sodium salt NaSH is used here as a source for HS•Since the first pKa of H2S is 6.8, at pH 7.2 the dominant species will be HS- with a fair
amount of H2S as well.
•Since H2S is a very volatile gas, some gas may have escaped during the course of the
experiment. This drives the equilibrium towards H2S
•Next step would be to add cystine to generate the persulfide chemically
No Reducing Agents
Acknowledgements
0
0
5
10
15
20
Time (min)
•Concentrations used are: 1mM DTT, 1mM Cystine, 1mM Cysteine, 90ug CSE, 5ug CD148,
30uM FDP. All wells are buffered with 50mM HEPES pH7.2.
•Dithiothreitol (DTT) is a commonly used reducing agent with no physiological relevance. It
is used in this experiment to determine the maximally reducible state of CD148 that may or
may not exist physiologically.
•CD148, only when reduced, acts upon the substrate FDP, generating fluorescence. Thus,
fluorescent intensity indicates active CD148.
•As shown above, CD148 regains activity only when exposed to the enzymatically produced
cysteine persulfide.
Dr. Katsuhiko Ono made recombinant CSE with GST tag.
Supported in part by NSF grant HRD-1302873.
References
1. Fukuto et. al. Small Molecule Signaling Agents: The Integrated Chemistry and Biochemistry of Nitrogen Oxides, Oxides of Carbon,
Dioxygen, Hydrogen Sulfide, and Their Derived Species. Chemical Research in Toxicology 25, 769-793. (2012).
2. Greiner, Romy, et al. "Polysulfides link H2S to protein thiol oxidation." Antioxidants & redox signaling 19.15 (2013): 1749-1765.
3. den Hertog et. al. Redox Regulation of Protein Tyrosine Phosphatases. Archives of Biochemistry and Biophysics 434, 11-15 (2005).
4. Ida, Tomoaki, et al. "Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling." Proceedings
of the National Academy of Sciences 111.21 (2014): 7606-7611.
5. Ono, Katsuhiko, et al. "Redox chemistry and chemical biology of H2S, hydropersulfides, and derived species: Implications of their
possible biological activity and utility." Free Radical Biology and Medicine 77 (2014): 82-94.
6. Williams M., Kwon J. T Cell Receptor Stimulation, Reactive Oxygen Species, and Cell Signaling. Free Radical Biology and
Medicine 37, 1144-1151 (2004).