Joan B. Mannick

S-nitrosylation of mitochondrial caspases

Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. In the current studies, we sought to identify the subpopulation of caspases that is regulated by S-nitrosylation. We report that the majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These studies suggest that S-nitrosylation plays an important role in regulating mitochondrial caspase function and that the S-nitrosylation state of a given protein depends on its subcellular localization.

Nitrosylation of Cytochrome c during Apoptosis

Cytochrome c released from mitochondria into the cytoplasm plays a critical role in many forms of apoptosis by stimulating apoptosome formation and subsequent caspase activation. However, the mechanisms regulating cytochrome capoptotic activity are not understood. Here we demonstrate that cytochrome c is nitrosylated on its heme iron during apoptosis. Nitrosylated cytochrome c is found predominantly in the cytoplasm in control cells. In contrast, when cytochrome c release from mitochondria is inhibited by overexpression of the anti-apoptotic proteins B cell lymphoma/leukemia (Bcl)-2 or Bcl-XL, nitrosylated cytochrome c is found in the mitochondria. These data suggest that during apoptosis, cytochrome c is nitrosylated in mitochondria and then rapidly released into the cytoplasm in the absence of Bcl-2 or Bcl-XL overexpression. In vitro nitrosylation of cytochrome c increases caspase-3 activation in cell lysates. Moreover, the inhibition of intracellular cytochromec nitrosylation is associated with a decrease in apoptosis, suggesting that cytochrome c nitrosylation is a proapoptotic modification. We conclude that nitrosylation of the heme iron of cytochrome c may be a novel mechanism of apoptosis regulation.

Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells

Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

NO means no and yes: regulation of cell signaling by protein nitrosylation.

Protein nitrosylation is emerging as a key mechanism by which nitric oxide regulates cell signaling. Nitrosylation is the binding of a NO group to a metal or thiol (-SH) on a peptide or protein. Like phosphorylation, nitrosylation is a precisely targeted and rapidly reversible posttranslational modification that allows cells to flexibly and specifically respond to changes in their environment. An increasing number of proteins have been identified whose activity is regulated by intracellular nitrosylation. This review focuses on proteins regulated by endogenous nitrosylation, the chemistry underlying nitrosylation, the specificity and reversibility of nitrosylation reactions, methods to detect protein nitrosylation, and the role of coordinated protein nitrosylation/denitrosylation in cell signaling.

Measurement of Protein S-Nitrosylation during Cell Signaling

S-Nitrosylation, the modification of a cysteine thiol by a nitric oxide (NO) group, has emerged as an important posttranslational modification of signaling proteins. An impediment to studying the regulation of cell signaling by S-nitrosylation has been the technical challenge of detecting endogenously S-nitrosylated proteins. Detection of S-nitrosylated proteins is difficult because the S-NO bond is labile and therefore can be lost or gained artifactually during sample preparation. Nevertheless, several methods have been developed to measure endogenous protein S-nitrosylation, including the biotin switch assay and the chemical reduction/chemiluminescence assay. This chapter describes these two methods and provides examples of how they have been used successfully to elucidate the role of protein S-nitrosylation in cell physiology and pathophysiology.

Analysis of Protein S‐Nitrosylation

S‐nitrosylation is the binding of an NO group to a cysteine or other thiol. Like phosphorylation, S‐nitrosylation is a precisely targeted and rapidly reversible post‐translational modification that serves as an on/off switch for protein function during cell signaling. However, unlike phosphorylation, S‐nitrosylation of proteins occurs nonenzymatically and is mediated, at least in part, by redox‐regulated chemical reactions in cells. Alterations in pH, pO2, cellular reductants, transition metals, and UV light lead to the loss and/or gain of S‐NO bonds. Due to the redox‐sensitive nature of the modification, analysis of protein S‐nitrosylation is technically difficult, since the S‐NO bond is easily disrupted during sample preparation. In addition, the level of S‐nitrosylated proteins in cells approaches the limit of detection of currently available technology. Despite these technical challenges, several useful methods have been developed recently to measure protein S‐nitrosylation in biological samples, and these are described in this unit.

UNIT 14.6 Analysis of Protein S-Nitrosylation

S‐nitrosylation is the binding of an NO group to a cysteine or other thiol. Like phosphorylation, S‐nitrosylation is a precisely targeted and rapidly reversible post‐translational modification that serves as an on/off switch for protein function during cell signaling. However, unlike phosphorylation, S‐nitrosylation of proteins occurs nonenzymatically, mediated, at least in part, by redox‐regulated chemical reactions in cells. Alterations in pH, pO2, cellular reductants, transition metals, and UV light lead to the loss and/or gain of S‐NO bonds. Due to the redox‐sensitive nature of the modification, analysis of protein S‐nitrosylation is technically difficult, the S‐NO bond formation being easily disrupted during sample preparation. In addition, the level of S‐nitrosylated proteins in cells approaches the limit of detection of currently available technology. Despite these technical challenges, several useful methods have been developed recently to measure protein S‐nitrosylation in biologic samples, and these are described in this unit.