1. Volume 156, Issue 6, Pages 1235-1246 (March 2014)
S-Glutathionylation of Cryptic Cysteines Enhances Titin Elasticity by Blocking Protein Folding 
Jorge Alegre-Cebollada, Pallav Kosuri, David Giganti, Edward Eckels, Jaime Andrés Rivas-Pardo, Nazha Hamdani, Chad M. Warren, R. John Solaro, Wolfgang A. Linke, Julio M. Fernández 
Cell 
Volume 156, Issue 6, Pages (March 2014)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Ig Domains in the I-Band of Titin Are Rich in Cryptic Cysteines
(A) Schematic representation of a half-sarcomere. Titin (in color) extends from the Z-disk to the M-band of the sarcomere. The approximate location of the I91 domain is indicated with an asterisk.
(B) Average cysteine content in the titin sequence.
(C) Structure of the titin I91 domain (also known as I27; PDB code 1TIT), highlighting in red its two cysteine residues.
(D) The cysteines in I91 are cryptic and can only be S-glutathionylated after unfolding. A SUMO-I91 construct was incubated with GSSG for 1 hr at different temperatures. S-glutathionylation was determined by western blotting and labeling with anti-glutathione antibodies (α-GSH). Ponceau S shows the total protein content.
See also Figure S1.
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4. Figure 2
S-Glutathionylation of Cryptic Cysteines Inhibits Refolding of I91
(A) I91 contains two cryptic cysteines that become exposed by mechanical unfolding. At a force of 130–175 pN, the unfolding of an I91 domain can be identified as a 25 nm step increase in end-to-end length. Exposure of the unfolded protein to GSSG leads to S-glutathionylation.
(B) A single polyprotein is unfolded at 175 pN in 100 mM GSSG and then held extended for a variable length of time (exposure time; 2 s top trace; 40 s bottom trace). The force is then quenched to zero for 5 s to allow the protein to refold. We measure the extent of refolding by applying a probe pulse.
(C) Refolded fraction as a function of exposure time for I918 polyproteins exposed to buffer only (downward triangles; n > 35), 100 mM GSSG (solid circles; n > 80), 200 mM GSH (upward triangles; n > 80) and refolded fraction in the presence of 100 mM GSSG for a mutant I918 polyprotein where all cysteine residues were replaced by alanines (ΔCys, squares; n > 120). The refolded fraction was calculated as the ratio between the number of unfolding steps detected during the probe pulse and the number of unfolding steps detected during the exposure pulse. Open symbols represent total refolding of I91 (weak + strong domains) in the presence of 100 mM GSSG, as determined using the double-probe pulse protocol in Figure 3. Error bars represent SEM.
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5. Figure 3 Graded Mechanical Modulation of I91 by S-Glutathionylation
(A) Experimental recordings showing unfolding steps during the probe pulse (open arrowheads). Each recording was obtained in 100 mM GSSG after a different exposure time followed by 5 s refolding. Refolded domains with different mechanical stability were detected using a two-level probe pulse (110 pN in blue followed by 175 pN in red).
(B) Refolded fraction (circles and squares; left axis) and failures (triangles; right axis) as a function of exposure time (n > 40). Squares indicate weak domains detected during the low-force regime; circles indicate native domains detected in the high-force regime. Data points at zero exposure time were obtained in buffer without GSSG (open symbols; n = 24). Solid lines were obtained from the kinetic model shown in Figure 5A. Error bars represent SEM.
See also Figure S2.
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6. Figure 4
S-Glutathionylation of Different Cysteines Produces Distinct Mechanical Phenotypes
(A) The single-cysteine mutant I91-Cys63 was mechanically unfolded and exposed to 100 mM GSSG for a variable amount of time. After a 5 s quench to allow for folding, refolding was probed using a linear force ramp up to 240 pN.
(B) Mechanical stability of refolded I91-Cys63 after varying exposure times to 100 mM GSSG (n > 25). Solid lines indicate fits of Gaussians with fixed widths and centroids.
(C) Refolded fraction of I91-Cys47 (triangles; n > 120) and I91-Cys63 (weak domains, squares; strong domains, circles; n > 150) at different exposure times for a fixed quench time of 5 s in 100 mM GSSG. Data points at zero exposure time were obtained in the absence of GSSG (open symbols; n > 150). Reaction rates of S-glutathionylation were measured from exponential fits (solid lines). Error bars represent SEM.
See also Figure S3.
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7. Figure 5
Kinetic Model
(A) Scheme that includes all possible reaction pathways for S-glutathionylation and mechanical outcomes. Numbers in parentheses are the asymptotic folding fractions.
(B) Sequence flanking the two cysteines in I91. Negatively charged residues are labeled red; positively charged residues are labeled blue.
(C) The rate of S-glutathionylation of Cys63 depends linearly on the concentration of GSSG. Error bars represent SEM.
See also Figure S4.
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8. Figure 6
Glutaredoxin Reverses the Mechanical Effects of S-Glutathionylation
(A) I9132–75 is S-glutathionylated through cleavage of its disulfide by GSH. Unfolding of I9132–75 increases the protein length by 11 nm, whereas S-glutathionylation increases it further by 14 nm. Exposure-quench-probe pulse protocol probes the refolding of an I9132–75 polyprotein in the presence of 100 mM GSH. During the exposure pulse, three unfolding events are followed by three S-glutathionylation events (solid arrowheads). The force is then quenched to zero for 5 s. No refolding events are observed during the probe pulse.
(B) Recording showing the unfolding and S-glutathionylation of four I9132–75 domains in the presence of 100 mM GSH and 45 μM GRX. After a 5 s quench, refolding is apparent during the probe pulse (25 nm steps; open arrowheads).
(C) Diagram of unfolding and S-glutathionylation of I9132–75. Numbers indicate the associated step sizes detected in force-clamp.
(D) GRX-mediated deglutathionylation.
(E) Refolded fraction after a 5 s quench in 100 mM GSH with or without 45 μM GRX. A baseline for refolding was measured using prereduced I9132–75 polyprotein in the presence of 100 mM GSH (n > 90). Error bars represent SEM.
See also Figure S5.
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9. Figure 7
Modulation of Cardiomyocyte Elasticity by S-Glutathionylation of Cryptic Cysteines in Titin
(A) Single human cardiomyocytes were pulled to different sarcomere lengths, and the resulting passive force was measured (elasticity test). Elasticity was probed initially and after subsequent incubations with 10 mM GSSG, 10 mM GSH, and 1 mM DTT; all incubations were done at long sarcomere lengths (2.6–2.7 μm; overstretch) to induce unfolding of titin Ig domains. Incubation times are indicated. Top trace shows the stretch protocol; bottom trace shows the resulting average passive tension generated by cardiomyocytes. Results are the average of at least eight cells. Insets show magnification of the relaxation phase after stretching the cardiomyocytes to SL = 2.4 μm.
(B) Mean peak force measured at various SLs. GSSG slack is an experiment where incubation with GSSG was done in relaxed cardiomyocytes (SL = 1.8 μm). Forces are normalized to the force measured at 2.4 μm SL in control conditions. Error bars represent SD. Inset: scheme of the experimental setup.
(C) Model of regulation of muscle elasticity through S-glutathionylation of cryptic cysteines in titin.
See also Figure S6.
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10. Figure S1 S-Glutathionylation of Titin, Related to Figure 1
(A) Cardiac proteins from mouse were subjected to SDS vertical agarose electrophoresis, and then transferred to PVDF membranes. Membranes were probed first with anti-glutathione antibody, stripped, and then reprobed with anti-titin antibody (9D10). The membrane on the right was stripped again and reprobed with an anti-myosin heavy chain antibody (MF20). Bands positive for anti-glutathione and titin antibodies are identified in two independent experiments. Specific recognition by the anti-glutathione antibody is demonstrated by the membrane on the right, where samples in the presence of β-mercaptoethanol (β-me) were also included. Incubation with β-mercaptoethanol prevents labeling of titin with the anti-glutathione antibody. Asterisk indicates one unidentified band. The membrane on the right was stained with Ponceau-S to show total protein content.
(B) Homology models of some of the Ig domains that contain cryptic cysteine residues. The numerous cryptic cysteines found in the titin I-band are possible targets for posttranslational modifications. Numbers in parentheses indicate corresponding amino acid boundaries in the sequence of canonical titin (Uniprot code: Q8WZ42).
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11. Figure S2
Force-Ramp Experiments Detect a Population of Refolded I91 Domains with Lower Mechanical Stability after Exposure to GSSG, Related to Figure 3
(A) To explore the possibility that S-glutathionylated I91 proteins could fold, although to a state less mechanically stable than the native state, we did refolding experiments with I91 in the presence of 100 mM GSSG. After a 5 s quench, folded domains were probed by a force ramp. During the ramp, the force was increased linearly from 0 to 200 pN at a rate of 40 pN/s. Folded domains were detected as 25-nm steps in length. In the absence of GSSG and in the presence of GSSG for short exposure times (2 s), domains unfold at high forces close to 200 pN. As the exposure time to GSSG increases (10 s, 20 s), fewer domains refold and the domains that do refold then unfold at a lower force (∼100 pN). At longer exposure times (40 s), refolding is rare.
(B) Histogram of the forces at which domains unfolded, as detected in the probe pulse. Two distinct populations are seen, with different mechanical stabilities (indicated by one and two asterisks respectively). For reference, one experimental trace is shown aligned with the histogram. N ≥ 40 for all conditions except for 40 s, where N = 13.
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12. Figure S3
Mechanical Stability of the S-Glutathionylated Single-Cysteine I91 Mutants and Folding Rate of Glutathionylated I91-Cys63, Related to Figure 4
(A) The mechanical stability of refolded I91-Cys47 in the absence (top histogram; N = 85) and in the presence of GSSG (bottom histogram, exposure time > 40 s; N = 40), measured using a force-ramp probe pulse (Figure 4A). No difference in mechanical stability was apparent upon S-glutathionylation of Cys47.
(B) Mechanical stability of refolded I91-Cys63 after 2 and 20 s exposure to 100 mM GSSG (N > 45). Solid lines indicate fits of Gaussians with fixed widths and centroids. The data from these fits were used to calculate the rate of S-glutathionylation at Cys63 (Figure 4C).
(C) Histograms show the distribution of unfolding forces of I91-Cys63 in the absence and presence of GSSG (40 s exposure). For both conditions, we plot the probability density of unfolding forces in a ramp for a two-state unfolding model, which depends on the parameters α0 (the unfolding rate at zero force) and Δx (the distance to the transition state) (Schlierf et al., 2004). For the condition in the absence of GSSG, we used the values for α0 and Δx for I91 WT (Li et al., 2002). If we assume that Δx is not affected by S-glutathionylation, we can reproduce the range of unfolding forces by using α0 = 0.01 s-1, which is 125 times higher than for the unmodified protein.
(D) Refolded fraction at different quench times for I91-Cys63 in the absence of GSSG (circles, N > 45) or after 20 s exposure to 100 mM GSSG (triangles, N > 110). Solid lines are exponential fits to the data. Folding rates are not apparently different, but the amplitude of refolding is decreased in the S-glutathionylated domain. Error bars represent SEM. We did not measure folding kinetics of S-glutathionylated I91-Cys47. Since the rate of glutathionylation at Cys47 is slow (Figure 5A), we would need to use exposure times over 100 s to ensure reaction is complete, making the experiment impractical.
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13. Figure S4
Rate of S-Glutathionylation of Cys63 in 10 mM GSSG, Related to Figure 5
(A) Refolding of I91-Cys63 in the presence of 10 mM GSSG was probed using the force-ramp protocol as in Figure 4A. Histograms show the mechanical stability of the refolded domains at different exposure times (N > 40). Solid lines are Gaussians fits with fixed widths and centroids.
(B) Refolded fraction of I91-Cys63 (weak domains, squares; strong domains, circles; N > 85) at different exposure times for a fixed quench time of 5 s in 10 mM GSSG. The proportion of weak and strong domains was calculated from the Gaussian fits in (A). Data points at zero exposure time were obtained in the absence of GSSG (open symbols; N > 150). Reaction rates of S-glutathionylation were measured from exponential fits (solid lines). For the fit to the weak domains data, we held fixed the value of the asymptote obtained at 100 mM GSSG (0.28, Figure 4C). Error bars represent SEM.
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14. Figure S5
Mechanical Stability of Refolded I9132–75 Domains and Disulfide-Reducing Activity of GRX, Related to Figure 6
(A) When pulling from oxidized I domains in the absence of GSH, only 11 nm steps (solid arrowheads) are detected in the first pulse, marking the unfolding of oxidized I The protein refolds efficiently.
(B) In the presence of 100 mM GSH, S-glutathionylation events are detected as steps at of 14 nm (diamonds). In this case, refolding events are identified as 25 nm steps (open arrowheads).
(C) The few domains that refold after GSH-mediated cleavage of the disulfide (bottom) show similar unfolding forces to domains refolded in the absence of GSH (top), showing that S-glutathionylation of I does not lead to formation of weakened domains.
(D) The rate of direct disulfide cleavage by glutaredoxin is negligible under the concentrations used. Extension recordings of GRX-mediated cleavage of I were extracted, summed, and averaged, for an experiment conducted in 180 μM GRX. The resulting trace (red) was fit with a single exponential (black), from which the first-order rate constant of direct disulfide cleavage by GRX was found to be  μM−1s−1. This leads to a rate of less than 2% as compared to the rate of disulfide cleavage by GSH under the experimental conditions used in Figure 6.
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15. Figure S6
S-Glutathionylation of Cryptic Cysteines in Titin Regulates Elasticity, Related to Figure 7
(A) When cardiomyocytes are incubated with 10 mM GSSG at slack SL that prevents Ig domain unfolding, no large-scale changes in elasticity are detected (N = 8).
(B) Periodic force protocol employed in the Monte Carlo simulations.
(C) Length response of titin to the applied force during the first cycle and after 12 hr of simulated stretch-and-relax. Both curves overlap showing that the stiffness is constant in the absence of glutathione. Stiffness was measured as the slope of the curve at 5 pN (dotted line).
(D) Addition of oxidized glutathione (100 mM) leads to a time-dependent decrease in stiffness. These effects stabilize after a few hours.
(E) Replacing the oxidized glutathione with an equal concentration of reduced glutathione reverses the effects in ∼1 hr.
(F) Titin stiffness at 5 pN as a function of time. At t = 6 hr, 100 mM glutathione in various states of oxidation was added to the simulated system. Different GSSG:GSH ratios are represented by different colors (%GSSG, from top: 0, 20, 50, 70, 80, 90, 100).
(G) Steady-state stiffness of titin in a 100 mM glutathione buffer at various redox potentials of the glutathione buffer. Error bars indicate SD, which were measured from the stable part of traces such as those shown in (F).
(H) Schematic diagrams of the sarcomere with titin in a state of (i) low, (ii) medium and (iii) high level of S-glutathionylation.
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