PMC

(A) [¹H-¹⁵N] TROSY NMR spectra of native and S-nitrosylated XIAP-RING domain. Chemical shift (CS) differences are shown between native RING (RING; blue) and S-nitrosylated RING (SNO-RING; red). Cross peaks with significant CS changes caused by S-nitrosylation (>0.02 ppm) are labeled with one letter amino-acid codes.
(B) CS perturbations induced by S-nitrosylation versus amino acid sequence.
(C) Higher-powered views of cross peaks with significant CS changes caused by S-nitrosylation (>0.02 ppm). Cross peaks are marked in either red (S-nitrosylated form) or blue (native form). Cross peaks without significant CS changes (<0.02 ppm) are labeled with black one letter amino-acid codes.
(D) NMR structure of XIAP-RING domain (PDB: 2ECG). Residues manifesting CS changes after S-nitrosylation are displayed in red (>0.02 ppm). Two cysteines (C450 and C471) located proximate to the chemically shifted/perturbed residues are colored yellow.

(A) SNO-XIAP was detected in cultured rat cerebrocortical neurons after exposure to NMDA by NO-biotin switch analysis. The NOS inhibitor NNA inhibited the formation of SNO-XIAP.
(B, C) Reduction of XIAP levels by shRNA sensitizes cortical neurons to NMDA. Cortical neurons were transfected with shRNAs, exposed to NMDA, and after 6 hr immunostained with anti-NeuN (to identify neurons) and anti-cleaved caspase-3 (to detect active caspase-3). The percentage of active caspase-3-positive neurons increased in XIAP shRNA-transfected neurons compared to control neurons (B). In parallel, the number of apoptotic neurons increased 8–14 hr later (C).
(D) Enhanced antiapoptotic activity of XIAP(C450H). Cortical neurons were transfected with XIAP or XIAP(C450H), exposed to NMDA, and after 12 hr immunostained with anti-NeuN. Apoptotic cell death was monitored by counting the number of Hoechst-stained nuclei containing condensed/fragmented chromatin (n ≥ 500 neurons scored in 3 independent experiments; *p < 0.05, **p < 0.01 by ANOVA). Data are presented as mean + SEM.

(A) SNO-caspase-3 transnitrosylates XIAP but not vice-versa. Transnitrosylation reactions were performed as described in the . Amounts (Input) of XIAP and caspase-3 were verified in each reaction. S-Nitrosylated proteins were detected by the NO-biotin switch assay. All panels depicted are from the same gel.
(B) SNO-caspase-3 does not transnitrosylate an XIAP mutant lacking the caspase-3 binding motif [XIAP(D148A)].
(C) Cleaved SNO-caspase-3 transnitrosylates XIAP. Caspase cleavage in HEK 293T lysates was activated with dATP and cytochrome c (dATP/CytC). Caspase-3 was then immunoprecipitated and exposed to SNOC. The resulting SNO-capase-3 was then incubated with recombinant XIAP to test for possible transnitrosylation. S-Nitrosylated proteins were detected by the NO-biotin switch assay and revealed transnitrosylation from cleaved (rather than full-length) SNO-caspase-3 to XIAP.
(D) Schematic Illustration of the Mechanism of SNO-XIAP—Mediated Neuronal Cell Death. Pathway (1): Under normal conditions, XIAP efficiently blocks caspases. Additionally, XIAP serves as an E3 ligase that ubiquitinates caspases and thus targets caspases for proteasomal degradation. Pathway (2): Under nitrosative conditions, NO inactivates the E3 ligase activity of XIAP via S-nitrosylation, thus stabilizing caspases, and sensitizing neurons to apoptotic stimuli. Pathway (3): Constitutively S-nitrosylated caspases serve as an additional mechanism to produce SNO-XIAP via transnitrosylation in neurons undergoing apoptotic cell death.

(A) In vitro S-nitrosylation of XIAP downregulates auto-ubiquitination. Purified recombinant GST-XIAP was incubated with SNOC (200 µM) or old SNOC, and 30 min later subjected to in vitro ubiquitination at RT. All samples were incubated with ATP. SNOC decreased XIAP auto-ubiquitination, as detected by anti-XIAP antibody.
(B) S-Nitrosylation of XIAP reduces auto-ubiquitination in HEK293T cells. HEK 293T cells co-transfected with HA-tagged ubiquitin plus Myc-tagged XIAP or Myc-tagged XIAPΔRING were incubated with SNOC to S-nitrosylate XIAP. After 2 to 12 hr, cell lysates were subjected to immunoprecipitation with anti-myc followed by immunoblot analysis with anti-HA to detect ubiquitinated proteins.
(C, D) S-Nitrosylation of XIAP in vitro down-regulates caspase-3 and -9 ubiquitination. Recombinant GST-XIAP was incubated with SNOC or old SNOC and then subjected to in vitro ubiquitination for 90 min at RT. All samples were incubated with ubiquitin and ATP. High molecular weight forms of caspases-3 and -9, apparently representing polyubiquitinated proteins, were detected with specific antibodies against these caspases.
(E) S-Nitrosylation of XIAP reduces caspase-3 ubiquitination in HEK293T cells. HEK293T cells co-transfected with HA-tagged ubiquitin, Fas, Myc-tagged mutant caspase-3(C285A), and His-tagged XIAP were exposed to SNOC. After 6 hr, cell lysates were immunoprecipitated with anti-myc antibody followed by immunoblot analysis with the indicated antibodies. Arrowhead, procaspase-3; *, immunoglobulin light chain.
(F) S-Nitrosylation of XIAP reduces caspase-9 ubiquitination in HEK293T cells. HEK 293T cells co-transfected with HA-tagged ubiquitin, Bax, FLAG-tagged mutant caspase-9(C285A), and Myc-tagged XIAP were exposed to SNOC. After 6 hr, cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblot analysis with the indicated antibodies. Arrowhead, XIAP; *, immunoglobulin heavy chain.

(A) S-Nitrosylation of XIAP at Cys450 by NO-biotin switch assay. HEK-nNOS cells were transfected with WT, C450H, or C471H XIAP constructs and exposed to the Ca²⁺ ionophore A23187 (5 µM) to activate endogenous nNOS.
(B) ETD-MS/MS spectra of XIAP-RING domain. To obtain MS/MS spectra, charge +6 precursors ([M+6H]⁺⁶) of unmodified RING (RING, top; 1053.68 m/z) and, after exposure to 10 µM SNOC, S-nitrosylated RING (SNO-RING, bottom; 1058.40 m/z) were isolated for the ETD experiment by a linear ion trap (LTQ) analyzer. Amino-acid sequence of the XIAP-RING domain is listed at the top of the RING spectrum. c and z ions present in both modified and unmodified proteins are indicated in blue, while ions detected only in the SNO-RING are in red. m/z values for C₁₁⁺³,C₁₃⁺², and C₁₆⁺³ ions in the SNO-RING spectrum are 425.17, 755.36, and 632.17, respectively. Additional MS data (LTQ Orbitrap XL-MS and ETD-MS/MS) are available in .
(C) SNO-XIAP in brains of patients with neurodegenerative diseases. Postmortem brain tissues from patients with neurodegenerative and non-CNS conditions (controls) were subjected to the NO-biotin switch assay. Images separated by a black line are from the same gel.

(A) S-Nitrosylation of XIAP by endogenous NO. HEK-nNOS cells were exposed to 5 µM A23187 to activate nNOS in the presence or absence of the NOS inhibitor, Nω-nitro-l-arginine (NNA), and SNO-XIAP was assessed by NO-biotin switch assay.
(B) Endogenous NO stabilizes cleaved caspase-3. HEK-nNOS cells co-transfected with myc-tagged caspase-3, myc-tagged XIAP or XIAPΔRING, and Bax were incubated with A23187 for 6 hr. Active caspase-3 (mycCaspase-3) was assessed by immunoblotting with anti-myc antibody. Endogenous c-myc detected by anti-myc antibody served as a loading control. SF, small fragment of cleaved caspase-3.
(C) NO reduces the inhibitory effect of XIAP on caspase activity. SH-SY5Y cells were transfected with a Bax expression plasmid. After 17 hr, cells were exposed to SNOC, and DEVDase activity was measured 7 hr later. Co-expression of XIAP attenuated Bax-induced caspase activation, an effect reversed by SNOC. XIAPΔR was less efficient than XIAP in inhibiting caspase activity triggered by Bax expression, and SNOC had no effect on XIAPΔR. As a control, the caspase inhibitor zVAD-fmk (25 µM) completely blocked Bax-induced caspase activation (n = 3–7).
(D) NO reduces the antiapoptotic effect of XIAP. A Bax expression plasmid was transfected into SH-SY5Y cells along with the indicated XIAP constructs. Apoptotic cell death was monitored by counting the number of nuclei with condensed or fragmented chromatin with Hoechst staining. Co-expression of XIAP attenuated Bax-induced apoptosis, an effect reversed by SNOC. XIAPΔR was less efficient than XIAP in protecting cells from cell death triggered by Bax expression, and SNOC had no effect on XIAPΔR. zVAD.fmk (25 µM) completely blocked Bax-induced cell death (n ≥ 3; *p < 0.01 by ANOVA). Data are presented as mean + SEM.

(A) Recombinant protein GST-XIAP (0.5 µM) was incubated with the physiological NO donor SNOC (200 µM) at RT. After 30 min, S-nitrosylated XIAP was assessed by release of NO, causing conversion of 2,3-diaminonaphthalene (DAN) to the fluorescent compound 2,3-naphthotriazole (NAT). The degree of NAT fluorescence from GST-XIAP was set at 100% (n = 3–4; *p < 0.001 by ANOVA).
(B) S-Nitrosylation of XIAP-RING domain in vitro. Recombinant truncated XIAP proteins, His-BIR2 (BIR2), His-BIR2-3 (BIR2-3), and His-BIR2-3-RING (BIR2-3-RING) (0.2 µM each) were incubated with SNOC. Thirty minutes later, S-nitrosylated protein was assessed by fluorescence assay. The degree of NAT fluorescence from the BIR-2-3-RING domains was set at 100% (n = 3–5; *p < 0.01).
(C) S-Nitrosylation of XIAP in intact cells. SH-SY5Y cells were exposed to 200 µM SNOC, and S-nitrosylated XIAP (SNO-XIAP) was detected by the NO-biotin switch assay. Equal amounts of total XIAP in the cell lysate (Input) were verified. SNO-XIAP was detected by the NO-biotin switch assay (Eluate). The “Control” sample was subjected to decayed (old) SNOC under the same conditions. MMTS, methyl methane thiosulfonate; w/o Ascorbate, w/o MMTS, and w/o Biotin represent controls without reducing agent, thiol blocking agent, or biotin linker, respectively; arrowhead, XIAP; *, 60 kDa.
(D) S-Nitrosylation of XIAP-RING domain in intact cells. HEK293T cells were transfected with GFP-tagged WT-XIAP or XIAPΔRING (ΔRING) and exposed to SNOC. Control WT was exposed to decayed SNOC. S-Nitrosylation of transfected XIAP (SNO-GFP-XIAP) was detected with the NO-biotin switch assay; equal loading was confirmed (Input GFP-XIAP). Data are presented as mean + SEM.