Nitric oxide (NO) is a putative signal molecule that is implicated in both physiological and pathological processes. Its bioactivity is mediated by S-nitrosylation, a ubiquitous redox-related modification of cysteine thiols. Dysregulated S-nitrosylation may play a causal role in a spectrum of human diseases including endotoxic shock and neurodegenerative diseases.

Prof Chen’s research group investigates the mechanism and cellular effects of redox-based post-translational modification of proteins, particularly focusing on S-nitrosylation. One line of their investigations is how S-nitrosothiol (SNO) affects intracellular events, such as protein localization and protein-protein interactions, and how NO and S-nitrosothiol (SNO) affect apoptosis and cell differentiation cascades. Their recent Journal of Cell Science paper1 addresses some of these questions, showing that the karyopherin chromosomal region maintenance 1 (CRM1) protein, the major receptor involved in classical nuclear protein export, is negatively regulated by S-nitrosylation under nitrosative stress.

Although CRM1 is considered to be a crucial gate-keeper in controlling nucleocytosolic movement of numerous signaling components and transcriptional regulators, it was unclear whether the activity of CRM1 could be regulated by signaling. CRM1 contains multiple evolutionarily-conserved cysteines, suggesting that its function could be redox-sensitive and may be regulated by S-nitrosylation. Intracellular NO, possibly in its SNO form, interacts with susceptible cysteine residues resulting in S-nitrosylation which then modulates cell signaling, transcription and enzyme activity. NO has been implicated in regulating the nucleocytoplasmic trafficking of a number of proteins, but it has been unclear whether it can modulate the nuclear transport machinery.

By exposing cellular CRM1 to extensive endogenous or exogenous nitric oxide (NO), Prof Chen’s group showed that it becomes S-nitrosylated, resulting in the loss of CRM1 interactions with nuclear export signals (NES) and repressing classical protein export. They found that CRM1 was extensively S-nitrosylated under nitrosative stress, and that S-nitrosylation of CRM1 at the C528 and C585 residues compromised its ability to recognize classical NESs. Since this facilitates transcription by the antioxidant response transcription factor Nrf2, Prof Chen and colleagues speculated that S-nitrosylation-mediated deactivation of CRM1 counteracts nitrosative stress-induced cell death.

This study has demonstrated that increasing cellular antioxidant potential is an important cellular defense mechanism in the presence of increased levels of reactive nitrogen species (RNS). Since NO is connected to multiple pathological processes such as endotoxic shock and neurodegenerative diseases, these findings have potential to yield therapeutic strategies to fight these diseases as they improve our understanding of how nitrosative stress affects nuclear transport.

Nitric oxide represses CRM1-dependent classical nuclear export. (A) HEK293 cells expressing Rev1.4+NES-GFP were untreated (Ctrl) or treated with LMB (5 ng/ml) for 20 minutes or with GSNO (1 mM) or H₂O₂ (1 mM) for 4 hours. Images of the GFP fluorescence are shown. (B) HEK293 cells expressing MoKA(NES2)-GFP (left panels) or MoKA(NES3)-GFP (right panels) were treated with GSNO or LMB for 4 hours. The GFP fluorescence was then examined. (C) RAW264.7 cells transiently expressing Rev1.4+NES-GFP were treated with LPS (100 ng/ml), LPS+SMT (100 mM) or PMA (0.5 mM) for 24 hours, or with GSNO (1 mM) for 4 hours, and the GFP fluorescence examined. Arrows denote the nuclei of transfected cells. (D) Nitrite concentration in the medium of samples in panel C; error bars denote s.d. Scale bars: 10 mm.

 

1Peng Wang, Guang-Hui Liu, Kaiyuan Wu, Jing Qu, Bo Huang, Xu Zhang, XiXi Zhou,

Larry Gerace and Chang Chen (2009) Repression of classical nuclear export by

S-nitrosylation of CRM1. Journal of Cell Science 122, 3772-3779.