The S-Nitrosylation Status of PCNA Localized in Cytosol Impacts the Apoptotic Pathway in a Parkinson’s Disease Paradigm

Abstract

It is generally accepted that nitric oxide (NO) or its derivatives, reactive nitrogen species (RNS), are involved in the development of Parkinson’s disease (PD). Recently, emerging evidence in the study of PD has indicated that protein S-nitrosylation triggers the signaling changes in neurons. In this study, SH-SY5Y cells treated with rotenone were used as a model of neuronal death in PD. The treated cells underwent significant apoptosis, which was accompanied by an increase in intracellular NO in a rotenone dose-dependent manner. The CyDye switch approach was employed to screen for changes in S-nitrosylated (SNO) proteins in response to the rotenone treatment. Seven proteins with increased S-nitrosylation were identified in the treated SH-SY5Y cells, which included proliferating cell nuclear antigen (PCNA). Although PCNA is generally located in the nucleus and participates in DNA replication and repair, significant PCNA was identified in the SH-SY5Y cytosol. Using immunoprecipitation and pull-down approaches, PCNA was found to interact with caspase-9; using mass spectrometry, the two cysteine residues PCNA-Cys81 and -Cys162 were identified as candidate S-nitrosylated residues. In addition, the evidence obtained from in vitro and the cell model studies indicated that the S-nitrosylation of PCNA-Cys81 affected the interaction between PCNA and caspase-9. Furthermore, the interaction of PCNA and caspase-9 partially blocked caspase-9 activation, indicating that the S-nitrosylation of cytosolic PCNA may be a mediator of the apoptotic pathway.

PCNA-interacting peptides reduce Akt phosphorylation and TLR-mediated cytokine secretion suggesting a role of PCNA in cellular signaling

“…A platform role of PCNA could also facilitate interaction between several proteins within PCNA-complexes. Nuclear PCNAcomplexes are dynamic and regulated by PTMs during genotoxic stress [2] and we hypothesize similar regulation of PCNA-interactions in cytosol in agreement with recent data [5].…”

“…These effects could be due to reduced protein stability of APIM-containing proteins when not interacting with PCNA, e.g. as detected here for PACT and previously shown for procaspases [3,5]. A platform role of PCNA could also facilitate interaction between several proteins within PCNA-complexes.…”

“…We have shown that high levels of cytosolic PCNA are found in multiple myeloma cells, and that targeting PCNA with APIM-peptide induces rapid caspase activation (caspases 3,7, 8 and 9) followed by apoptosis in multiple myeloma, but not in healthy primary cells [4]. Recently, cytosolic PCNA was shown to bind and stabilize procaspase-9 in a neuroblastoma cell line, and interestingly S-nitrosylation of PCNA blocked this interaction suggesting that the binding is regulated by posttranslational modifications (PTMs) [5]. Furthermore, PCNA on the cell surface of cancer cells interacts with the natural cytotoxicity receptor NKp44 on activated natural killer cells and inhibits their cytotoxic function.…”

Mitochondrial dysfunction and cell death in neurodegenerative diseases through nitroxidative stress

“…Suppression of these mitochondrial complex proteins leads to impairment of the mitochondrial ETC, resulting in insufficient electron transfer and greater ROS leakage from the respiratory chain. Cytosolic caspases can be also activated by S-nitrosylated proliferating nuclear cell antigen (PCNA)and thus activated (Yin et al, 2015), thus resulting in elevated apoptosis of neuronal cells. Consequently, we expect to see markedly increased oxidative stress, lipid peroxidation and decreased energy production following oxidative modifications of many proteins, as reported in neurodegenerative diseases.…”

“…Thus subsequent NAD + depletion and mitochondrial permeability transition (MPT) are sequential and necessary steps in PARP-1-mediated cell death (Alano et al, 2004; Alano et al, 2010; Lu et al, 2014). Furthermore, many other laboratories reported the importance of S-nitrosylation and/or nitrated proteins such as parkin, GAPDH, XIAP, Prx2, PDI and caspase 3 in neurological diseases (Butterfield et al, 2010, 2014; Cho et al, 2009; Fang et al, 2007; Kwak et al, 2010; Walker et al, 2010; Yin et al, 2015). However, it is likely that additional proteins in the brain could have been nitrated and/or S-nitrosylated under increased nitroxidative stress after exposure to neurotoxic agents or in neurological conditions.…”