S-nitrosylation in Neuropathic pain and Autophagy

Dissertation for attaining the PhD degree of Natural Sciences submitted to the Faculty of Biochemistry, Chemistry and Pharmacy of the Johann Wolfgang Goethe University in Frankfurt am MainNeuropathic pain is a maladaptive form of chronic pain caused by a primary injury or lesions in the central or peripheral nervous systems. Recently nitric oxide (NO) has emerged as important pro-nociceptive signaling molecule in pain signaling and processing. Chemical inhibiton or deletion of Nitric oxide synthase (NOS), as well and inhibition of the NOS-coenzyme tetrahydrobiopterin is known to reduce or inhibit neuropathic pain. NO exert its influence through two major pathways: by stimulation of sGC and by direct S-nitrosylation (SNO) of target proteins. This study assessed in the spinal cord the SNO-proteome with two methods, two-dimensional S-nitrosothiol difference gel electrophoresis (2D SNO-DIGE) and SNO-site identification (SNOSID) at baseline and 24 h after sciatic nerve injury with/without pretreatment with the nitric oxide synthase inhibitor L-NAME. At 24h after nerve injury, SNO-DIGE revealed 30 proteins with increased S-nitrosylation and 23 proteins with decreased S-nitrosylation. SNO-sites were identified for 17 out of these 53 proteins. L-NAME pretreatment substantially reduced both constitutive and nerve injury evoked up-S-nitrosylation. For the top candidates S-nitrosylation was confirmed with the biotin switch technique and time course analyses at 1 and 7 days after nerve injury showed that SNO modifications of protein disulfide isomerase (PDI), glutathione synthase (GSS) and peroxiredoxin-6 (Prdx6) had returned to baseline within 7 days whereas S-nitrosylation of mitochondrial aconitase 2 (Aco2) was further increased. The identified SNO modified proteins are primarily involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. Several targets, including PDI, Heat shock cognate 71 kDa protein, and Serpin B6, indicated that NO might play a role in protein quality control, metabolism, and folding. Subsequently an investigation into the potential role of NO and SNO in proteasomal degradation and autophagy was performed. Autophagy is a basic catabolic mechanism involving the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery and is an important factor in the recovery of neurons after injury. A cellular model of neuronal nitric oxide synthase (nNOS) over-expressing neuroblastoma cell culture stimulated with rapamycin to induce autophagy was used. The effects of nNOS overexpression on autophagic processes were evaluated by western blotting with antibodies for known markers of autophagy. S-nitrosylation was evaluated using a combination of SNO-DIGE, SNOSID, SNO-SILAC, and SNO-ELISA methods. Increased (+ 144%, p < 0.05) LC3-I / LC3-II ratio in the nNOS over-expressing cells compared to the wild type after stimulation with rapamycin suggested that the autophagic activity may be impaired by the increase of NO and possibly by an increase of direct protein S-nitrosylation. In the nNOS over-expressing cells the total ubiquitination was increased (+100%, p < 0.01) while it was decreased (-30%, p < 0.01)) in the wild type cells after rapamycin stimulation. This indicates that the targeting or degradation processes may be impaired by the increase in S-nitrosylation of the E1, E2 or E3 ubiquitin ligases. The results suggest that S-nitrosylation may regulate protein folding, ubiquitination, and possibly de novo protein generation. In this study several proteins have emerged as major candidates for being targets of S-nitrosylation and consequentially may affect the autophagic processes, namely heat shock cognate 71 kDa protein and pyruvate kinase isozymes M1/M2, calreticulin, ubiquitin-conjugating enzyme E2 D1, and elongation factor 2. The results suggest that NO, in addition to its known regulation of cGMP signaling, may contribute to the fine tuning of protein folding and degradation which are key mechanisms for allowing neurons to recover stability after axonal injury

Biochemical characterization of a native group III trypsin ZT from Atlantic cod (Gadus morhua)

Publisher's version (útgefin grein)Atlantic cod trypsin ZT is biochemically characterized for the first time in this report in comparison to a group I trypsin (cod trypsin I). To our knowledge, trypsin ZT is the first thoroughly characterized group III trypsin. A more detailed understanding of trypsin ZT biochemistry may give insight into its physiological role as well as its potential use within the biotechnology sector. Stability is an important factor when it comes to practical applications of enzymes. Compared to trypsin I, trypsin ZT shows differences in pH and heat stability, sensitivity to inhibitors and sub-site substrate specificity as shown by multiplex substrate profiling analysis. Based on the analysis, trypsin ZT cleaved at arginine and lysine as other trypsins. Furthermore, trypsin ZT is better than trypsin I in cleaving peptides containing several consecutive positively charged residues. Lysine- and arginine-rich amino acid sequences are frequently found in human viral proteins. Thus, trypsin ZT may be effective in inactivating human and fish viruses implying a possible role for the enzyme in the natural defence of Atlantic cod. The results from this study can lead to multiple practical applications of trypsin ZT.This work was supported by the AVS R&D Fund of Ministry of Fisheries and Agriculture in Iceland [grant reference number: R15 046-15 , R069-08 , R11 028-11 and R14 044-14 ]; and Technology Development Fund (The Icelandic Centre for Research ) [grant reference number: 120852-0611 and 131804-0611 ]. The funding source had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.Peer Reviewe

S-nitrosylation in Neuropathic pain and Autophagy

Doktorsritgerð varin við Goethe háskólann í Frankfurt am Main sumarið 2013.Neuropathic pain is a maladaptive form of chronic pain caused by a primary injury or lesions in the central or peripheral nervous systems. Recently nitric oxide (NO) has emerged as important pro-nociceptive signaling molecule in pain signaling and processing. Chemical inhibiton or deletion of Nitric oxide synthase (NOS), as well and inhibition of the NOS-coenzyme tetrahydrobiopterin is known to reduce or inhibit neuropathic pain. NO exert its influence through two major pathways: by stimulation of sGC and by direct S-nitrosylation (SNO) of target proteins. This study assessed in the spinal cord the SNO-proteome with two methods, two-dimensional S-nitrosothiol difference gel electrophoresis (2D SNO-DIGE) and SNO-site identification (SNOSID) at baseline and 24 h after sciatic nerve injury with/without pretreatment with the nitric oxide synthase inhibitor L-NAME. At 24h after nerve injury, SNO-DIGE revealed 30 proteins with increased S-nitrosylation and 23 proteins with decreased S-nitrosylation. SNO-sites were identified for 17 out of these 53 proteins. L-NAME pretreatment substantially reduced both constitutive and nerve injury evoked up-S-nitrosylation. For the top candidates S-nitrosylation was confirmed with the biotin switch technique and time course analyses at 1 and 7 days after nerve injury showed that SNO modifications of protein disulfide isomerase (PDI), glutathione synthase (GSS) and peroxiredoxin-6 (Prdx6) had returned to baseline within 7 days whereas S-nitrosylation of mitochondrial aconitase 2 (Aco2) was further increased. The identified SNO modified proteins are primarily involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. Several targets, including PDI, Heat shock cognate 71 kDa protein, and Serpin B6, indicated that NO might play a role in protein quality control, metabolism, and folding. Subsequently an investigation into the potential role of NO and SNO in proteasomal degradation and autophagy was performed. Autophagy is a basic catabolic mechanism involving the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery and is an important factor in the recovery of neurons after injury. A cellular model of neuronal nitric oxide synthase (nNOS) over-expressing neuroblastoma cell culture stimulated with rapamycin to induce autophagy was used. The effects of nNOS overexpression on autophagic processes were evaluated by western blotting with antibodies for known markers of autophagy. S-nitrosylation was evaluated using a combination of SNO-DIGE, SNOSID, SNO-SILAC, and SNO-ELISA methods. Increased (+ 144%, p < 0.05) LC3-I / LC3-II ratio in the nNOS over-expressing cells compared to the wild type after stimulation with rapamycin suggested that the autophagic activity may be impaired by the increase of NO and possibly by an increase of direct protein S-nitrosylation. In the nNOS over-expressing cells the total ubiquitination was increased (+100%, p < 0.01) while it was decreased (-30%, p < 0.01)) in the wild type cells after rapamycin stimulation. This indicates that the targeting or degradation processes may be impaired by the increase in S-nitrosylation of the E1, E2 or E3 ubiquitin ligases. The results suggest that S-nitrosylation may regulate protein folding, ubiquitination, and possibly de novo protein generation. In this study several proteins have emerged as major candidates for being targets of S-nitrosylation and consequentially may affect the autophagic processes, namely heat shock cognate 71 kDa protein and pyruvate kinase isozymes M1/M2, calreticulin, ubiquitin-conjugating enzyme E2 D1, and elongation factor 2. The results suggest that NO, in addition to its known regulation of cGMP signaling, may contribute to the fine tuning of protein folding and degradation which are key mechanisms for allowing neurons to recover stability after axonal injury

Nitric oxide contributes to protein homeostasis by S-nitrosylations of the chaperone HSPA8 and the ubiquitin ligase UBE2D

Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine. HSPA8 is involved in protein folding, organelle import/export and chaperone-mediated LAMP2a-dependent autophagy (CMA). A set of deep redox and full proteome analyses, plus analysis of autophagy, CMA and ubiquitination with rapamycin and starvation as stimuli confirmed the initial observations and revealed a substantial increase of SNO modifications in nNOS+ cells, in particular targeting protein networks involved in protein catabolism, ubiquitination, carbohydrate metabolism and cell cycle control. Importantly, NO-independent reversible oxidations similarly occurred in both cell lines. Functionally, nNOS caused an accumulation of proteins, including CMA substrates and loss of LAMP2a. UBE2D activity and proteasome activity were impaired, resulting in dysregulations of cell cycle checkpoint proteins. The observed changes of protein degradation pathways caused an expansion of the cytoplasm, large lysosomes, slowing of the cell cycle and suppression of proliferation suggesting a switch of the phenotype towards aging, supported by downregulations of neuronal progenitor markers but increase of senescence-associated proteins. Hence, upregulation of nNOS in neuronal cells imposes aging by SNOing of key players of ubiquitination, chaperones and of substrate proteins leading to interference with crucial steps of protein homeostasis. Keywords: Redox modification, Nitric oxide, Autophagy, Ubiquitin, Chaperone, Lysosome, Posttranslational modification, Starvation, Rapamycin, Senescenc

Nitric oxide contributes to protein homeostasis by S-nitrosylations of the chaperone HSPA8 and the ubiquitin ligase UBE2D

Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine. HSPA8 is involved in protein folding, organelle import/export and chaperone-mediated LAMP2a-dependent autophagy (CMA). A set of deep redox and full proteome analyses, plus analysis of autophagy, CMA and ubiquitination with rapamycin and starvation as stimuli confirmed the initial observations and revealed a substantial increase of SNO modifications in nNOS+ cells, in particular targeting protein networks involved in protein catabolism, ubiquitination, carbohydrate metabolism and cell cycle control. Importantly, NO-independent reversible oxidations similarly occurred in both cell lines. Functionally, nNOS caused an accumulation of proteins, including CMA substrates and loss of LAMP2a. UBE2D activity and proteasome activity were impaired, resulting in dysregulations of cell cycle checkpoint proteins. The observed changes of protein degradation pathways caused an expansion of the cytoplasm, large lysosomes, slowing of the cell cycle and suppression of proliferation suggesting a switch of the phenotype towards aging, supported by downregulations of neuronal progenitor markers but increase of senescence-associated proteins. Hence, upregulation of nNOS in neuronal cells imposes aging by SNOing of key players of ubiquitination, chaperones and of substrate proteins leading to interference with crucial steps of protein homeostasis

Rab7-a novel redox target that modulates inflammatory pain processing.

Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity