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S-Nitrosylation of Matrix Metalloproteinases: Signaling Pathway to Neuronal Cell Death
Zezong Gu, Marcus Kaul, Boxu Yan, Steven J. Kridel, Jiankun Cui, Alex Strongin, Jeffrey W. Smith, Robert C. Liddington, and Stuart A. Lipton

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Fluorometric Detection of S-Nitrosothiols. The concentration of S-nitrosothiol formation was detected by conversion of the fluorescent compound 2,3-naphthyltriazole (NAT) from 2,3-diaminonaphthalene (DAN) at an emission wavelength of 360 nm and an excitation wavelength of 260 nm using a FluoroMax-2 spectrofluorometer and DataMax software (Instruments S.A., Inc., Edison, NJ). Conversion to S-nitrosothiol is linear over the range 0.05 to 50 Greek Letter MuM, as previously described (S1). NAT was prepared by dissolving 500 mg of DAN in 20 ml of glacial acetic acid, diluting the solution to 100 ml with distilled water on ice, then rapidly reacting it with 2.5 ml of 1.2 M NaNO2. The precipitated NAT was recrystallized three times from boiling water containing decolorizing carbon. The white needle-like crystal NAT had a melting point of 194 °C. The fluorescence intensity curve of serial NAT dilutions was used to construct a standard curve. Previously, it had been reported that MMPs were not activated by NO donors but only by peroxynitrite (ONOO-) (S2, S3); however, those studies did not monitor S-nitrosothiol formation immediately after exposure to specific endogenous nitrosylating agents, as performed here.

Peptide mass fingerprinting. Peptide mass fingerprinting was used as described previously to identify tryptic fragments of MMP-9 (S4). Mass spectra were obtained with a Voyager DE-RP MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen laser (337 nm, 3-ns pulse). Peptide Mass software on the ExPASy Molecular Biology Server at was used to calculate the mass and identity of each fragment. Results of digestion of human R-proMMP-9 by trypsin in solution after exposure to SNOC are presented in the printed text. For in-gel digestion (fig. S4), free cysteines were protected by iodoacetamide alkylation in the absence of SNOC to avoid cleavage followed by uncontrolled disulfide formation. Among eleven mass peaks, we observed seven signature masses of human MMP-9 fragments that were virtually identical ( <_0.1 variation="variation" to="to" those="those" predicted="predicted" from="from" theoretical="theoretical" tryptic="tryptic" fragments="fragments" of="of" mmp-9="mmp-9" deduced="deduced" the="the" published="published" amino="amino" acid="acid" sequences="sequences" fig.="fig." s4="s4" top="top" panel.="panel." one="one" these="these" peaks="peaks" represented="represented" region="region" responsible="responsible" for="for" cysteine="cysteine" switch="switch" in="in" propeptide="propeptide" domain="domain" prommp-9="prommp-9" cgvpdlgr="cgvpdlgr" _816="_816" da="da" that="that" had="had" been="been" alkylated="alkylated" with="with" iodoacetamide="iodoacetamide" _57="_57" yield="yield" a="a" molecular="molecular" mass="mass" _873.4="_873.4" acet-cgvpdlgr.="acet-cgvpdlgr." contrast="contrast" vitro="vitro" exposure="exposure" r-prommp-9="r-prommp-9" snoc="snoc" prior="prior" attempted="attempted" alkylation="alkylation" yielded="yielded" fragment="fragment" on="on" maldi-tof="maldi-tof" analysis="analysis" at="at" _848.3="_848.3" indicating="indicating" addition="addition" stable="stable" _32="_32" adduct="adduct" instead="instead" middle="middle" panel="panel" em="em">n = 4 experiments). Masking thiol groups by prior alkylation with iodoacetamide before exposure to SNOC (yielding a mass of 873.8 Da) blocked the addition of the 32 Da adduct (fig. S4, bottom panel). In this chemical context, therefore, the 32 Da adduct represented addition of two oxygen molecules to the cysteine residue to form a sulfinic acid derivative (SO2H-CGVPDLGR at 848 Da) (25).

Analysis of Neurotoxicity. In the experiments testing the neurotoxicity of MMPs in cerebrocortical cultures, we estimate that the neurons were exposed to picomolar levels of exogenous activated MMP-9 as follows. We calculated the difference between the amount of exogenous NO-activated MMP-9 added to a culture dish and that recovered after a short (5 min) incubation. We assumed that this difference in activity (minus baseline MMP activity) reflected the exogenous MMP-9 present on the cell surface that was responsible for the subsequent neuronal apoptosis that occurred over the ensuing 18 hours. We then multiplied this activity by 10% since from in situ zymography only ~10% of cells with MMP activity at their surface were neurons. The result was that neurons were exposed to ~150 pM exogenous NO-activated MMP-9.

Molecular Modeling. We modeled MMP-9 structure (fig. S5) using MODELLER software (Version 4) and the related human MMP-2 crystal structure (PDB code 1CK7) (8).

Supplemental Figure 1. Half-lives of S-nitroso-MMP-9 (Filled Circle Symbol, circles) and SNOC (Filled Triangle Symbol, triangles). NO released from S-nitroso-MMP-9 or SNOC was detected by NAT conversion from DAN. The half-lives of SNOC and S-nitroso-MMP-9 were <_10 min="min" and="and" _30="_30" respectively.="respectively." p="p">

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Supplemental Figure 2. Activation of the full-length human proMMP-9 in vitro by SNOC. proMMP (100 ng/ml, Chemicon, Temecula, CA) was reacted with 200 Greek Letter MuM SNOC at room temperature. MMP-9 activity was subsequently assessed by the cleavage rate of fluorogenic Substrate I Peptide (25 Greek Letter MuM, Calbiochem, San Diego, CA; excitation wavelength, 280 ± 1 nm; emission wavelength, 360 ± 5 nm). SNOC led to activation of full-length MMP-9 in a fashion similar to that observed with MMP-9 that lacked the hemopexin domain (Fig. 2C).

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Supplemental Figure 3. Exogenous MMP-9 activated by SNOC induces neuronal apoptosis in cerebrocortical cell culture. (A) Neurons exhibiting MMP activity were identified by in situ zymography with the substrate DQ-gel-FITC (green), in combination with immunocytochemical staining using anti-MAP-2 antibody (red) as a neuronal marker. Nuclear DNA was labeled with Hoechst 33342 (blue). (B) Apoptotic neurons were identified by staining with anti-MAP-2 (red) and TUNEL (green) in conjunction with nuclear morphology, as evaluated by DNA staining with Hoechst 33342 (blue). Scale bar, 20 Greek Letter Mum.

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Supplemental Figure 4. Peptide mass fingerprinting analysis of the modified thiol group of the cysteine residue within the highly conserved auto-inhibitory prodomain of human MMP-9. Top: R-proMMP-9 was subjected to SDS-PAGE under nonreducing conditions and visualized by Coomassie blue staining. The mass spectra from in-gel digested R-proMMP-9 revealed seven signature masses (arrows) from eleven tryptic fragments in the mass range of 700 to 1600 Da. The peak at 873.4 Da represents the peptide CGVPDLGR alkylated with iodoacetamide in the human prodomain fragment (acet-CGVPDLR). Middle: After exposure of R-proMMP-9 to 200 Greek Letter MuM SNOC in vitro, MALDI-TOF mass spectrometry revealed a peak at 848.3 Da, representing SO2H-CGVPDLGR. Bottom: Reduction of R-proMMP-9 with dithiothreitol (DTT) followed by alkylation with iodoacetamide prior to exposure to SNOC prevented the shift of the 873 Da peak representing acet-CGVPDLR.

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Supplemental Figure 5. Model of MMP-9 activation by S-nitrosylation and subsequent oxidation. (A) Molecular surface of a partial sequence of human MMP-9 (from 97Pro to 411His without the fibronectin repeats found between 216Val and 391Gln) (8, 24). Color coded by charge with positive charge in violet, negative charge in red; propeptide domain (97Pro to 106Arg) designated by a yellow ribbon; catalytic domain (401His to 411His) in green. In proMMP-9, Zn2+ is coordinated by a cysteine and three histidine residues. R98, C99, and E402 fit the proposed consensus motif for S-nitrosylation (20). (B, C) Proposed structure-based chemistry of NO-induced MMP-9 activation. Reactivity of the catalytic cysteine sulfur of MMP-9 appears to be enhanced by increased nucleophilicity of 402Glu (shown in red) to S-nitrosylating agents (SNOC = Cys-NO, for example). The sulfur bound at the zinc site appears to be highly nucleophilic, which may give high initial reactivity to NO from its endogenous donors. The S-nitroso-MMP-9 propeptide domain appears to be more easily broken up in this highly polar environment and replaced by a nucleophilic water molecule. Reaction with H2O of the S-nitrosothiol group forms sulfenic acid (-SOH), as observed in glutathione reductase (25, S5). The reversible sulfenic acid can serve as an intermediate leading to subsequent irreversible oxidation steps via reactive oxygen species to sulfinic (-SO2H) and sulfonic (-SO3H) acids. R, Arg; C, Cys; E, Glu; H, His.

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Supplemental References:

S1. D. A. Wink et al.,Meth. Enzymol.301, 201 (1999).

S2. T. Okamoto et al.,Archiv. Biochem. Biophys.342, 261 (1997).

S3. W. Eberhardt et al.,Kidney Intl.57, 59 (2000).

S4. B. Yan, J. W. Smith, J. Biol. Chem.275, 39964 (2000).

S5. K. Becker, S. N. Savvides, M. Keese, R. H. Schirmer, P. A. Karplus, Nature Struc. Biol.5, 267 (1998).