a) S-nitrosylation is a post-translational protein modification that may occur in the presence of excess free radical, nitric oxide, and thiol groups present on specific cysteine residues of some proteins. This modification has been established as part of stimulus evoked signaling mechanisms necessary for specific cell responses. Many studies have provided evidence for nitric oxide activating apoptosis via various mechanisms including activation of mitochondrial pathways that result in “release of cytochrome c and endonuclease G, as well as the inhibition of NF-κB (nuclear factor κB) and increased p53 expression”. (Feng) Researchers in the State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University, China, found that apoptosis is induced through inhibiting the phosphorylation of extracellular signal-regulated kinase (ERK) through S-nitrosylation of a cysteine residue—potentially Cys183—by intracellular nitric oxide. They first supported that NO reinforces cell apoptosis through treating cells with Sodium Nitroprusside (SNP), a nitric oxide donor, with and without the presence of an NO scavenger, hemoglobin (HB). (Figure 1) They then went on to examine whether or not nitric oxide had an effect on the phosphorylation of ERK due to its pathway being evident as a mechanism for promoting cell survival. The demonstrated that SNP treatment resulted in “significantly diminished” levels of phosphorylated ERK, and ERK substrate ELK1, after four to five hours. Using a constitutively active form of ERK (TEY-EED), they simulated a situation in which SNP treatment had no effect on the kinase activity of the already more effective
TEY-EED. Going on to measure the effects of ERK kinase activity on apoptosis showed that wild type ERK cells treated with SNP had “substantially increased apoptosis (from 9.38% to 56.94%) while transfectants with kinase active mutants (TEY-EED) partially reverse the apoptotic effect (from 56.94% to 20.47%)”. (Feng) Overall these data heavily supports the concept that decreased kinase activity of ERK from S-nitroslyation plays a key role in signaling for apoptosis. Systematic mutations of ERK cystine residue to alanine residues demonstrated that the principal cysteine residue affecting kinase activity from S-nitrosylation is most likely Cys183. Using a Cys183 mutant (C183A mutant), the researchers were able to prove that SNP did not effect the kinase activity of the ERK in C183A mutant cells which also resulted in decreased apoptosis. Overall, the researchers at Nanjing University, China proved that nitric oxide can act as a second messenger in apoptosis signaling pathway through S-nitrosylation of ERK.
a) The p53 protein, encoded by tumor suppressor gene TP53, has multiple regulatory functions within multicellular organisms. In more recent literature, is has been established that p53 exhibits redox sensitivity through 10, highly, redox-reactive cysteine residues that comprise its zinc-binding domain. Oxidation of these residues may result in decreased binding-affinity for DNA and a resulting change in p53’s functions as a transcription factor. In addition to p53’s redox sensitivity to ROS and other small redox molecules such as thioredoxin, p53 may be directly susceptible to oxidative damage that occurs in the DNA itself through the charge transport mechanism. Researchers at the California Institute of Technology provide a genomic redox sensing and signaling mechanism through which redox-sensitive transcription factors may be globally activated in response to genomic oxidative stress through DNA’s capability of charge transport: “long-range funneling of oxidative damage to sites of low oxidation potential”. (Genereux, Figure 3) Evidence has been provided for p53’s capacity for regulating intracellular redox homeostasis through mediated transcription of “multiple antioxidant molecules such as SESN1 and SESN2 and GPX1, which function to decrease ROS level and to promote cell survival”, (Trachootham) as well as transcription of other proteins that support tumor suppression pathways: apoptosis, senescence, and autophagy. (Vousden, Figure 4) Overall, p53 has demonstrated its capability of regulating cell repair and programmed cell death through its functions as a systematic transcription factor that can react accordingly to cellular stress of which “include[s] DNA damage, oncogene activation, telomere elimination, and hypoxia” through direct or protein mediated sensitivity. (Genereux)
b) Researchers at the California Institute of Technology have proposed a mechanism by which charge transport may regulate p53 activity under oxidative stress. (Augustyn) Furthermore, “with certain stimuli, such as oxidative stress and DNA damage, p53 is stabilized by posttranslational modification and translocates to the nucleus”. (Trachootham)  A phosphorylation—acetylation cascade may be triggered by histone acetyltransferases (HATs) at two separate sites which results in enhancement of sequence-specific DNA binding. (Sakaguchi)
c) Hypoxia inducible factor 1 (HIF-1) is a transcription factor that acts as a master regulator in mediating the cell’s response to hypoxia by inducing the transcription of multiple genes, including those responsible for tumor growth, angiogenesis, and metastasis pathways. Researchers at the Johns Hopkins University School of Medicine proved that the relationship between hypoxia and HIF-1 was causative by exposing cells to decreased, and a subsequent increase, of oxygen and measuring the expression of HIF-1α and HIF-1β. They established that after increasing oxygen, HIF-1α and HIF-1β RNA and protein levels “decayed rapidly [which is] consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia”. (Wang) Tissue- specific metabolic changes in the cell that occur in response to hypoxia are mediated by HIF-1, and include many complex processes such as, red-blood cell oxygen-transport capacity, angiogenesis, glucose metabolism, proliferation/survival, and apoptosis. Any abnormal HIF-1 expression has a high potential for generating disease states: higher expression resulting in cancer and low expression or knockouts resulting in abnormal embryonic development and decreased blood vessel formation. (Ke) The oxygen responsive subunit of HIF-1, HIF-1α, selectively interacts with the constitutively active subunit, HIF-1β, based on the concentration of oxygen in the cell. HIF-1α will escape proteasomal degradation, translocate to the nucleus, and dimerize with HIF-1β under low oxygen concentrations which results in the activation of HIF-1’s transcription factor functions. (Hielscher) Hypoxia-induced formation of reactive oxygen species is the main mechanism by which HIF-1α is stabilized and rescued from proteasomal degradation. Researchers have established a causative role for ROS in HIF-1α stabilization through experiments that include the depletion of mitochondria resulting in decreased ROS, suppressed expression of the Rieske iron-sulfur protein of complex III resulting in decreased ROS, each of which affected HIF-1α by causing attenuated stabilization. (Hielscher) Researchers at the Department of Basic Pharmaceutical Sciences, West Virginia University, used cadmium, in vitro and in vivo models, to investigate mechanisms of angiogenesis. They found that treatment with cadmium (CdCl2) “activated extracellular signal-regulated kinases (ERK) and AKT signaling and elevated the expression of a key downstream proangiogenic molecule hypoxia-inducible factor-1 (HIF-1)” (Jing) through increased presence of reactive oxygen species development based on inhibition mechanisms of ROS using catalase and diphenyleneiodonium chloride. Inhibition of ROS resulted in attenuated ERK, AKT, p70S6K1 activation, and HIF-1α expression; and analogous functions could be postulated from the results obtained doing these experiments on human bronchial epithelial cells. The researchers then went on to substantiate these results by measuring angiogenic ability in cadmium-treated BEAS-2B cells. “Tumor tissues formed by the cadmium-treated BEAS-2B cells developed significantly more blood vessels compared with the tissues in control group”. (Jing, Figure 5) These results confirm the role of cadmium in promotion of angiogenesis and vigorously imply mechanisms that associate reactive oxygen species, HIF-1, and the promotion of angiogenesis. (Jing)
a) Metabolic syndrome results in an increased change of heart disease, stroke, and diabetes based on risk factors such as increased blood pressure, a high blood sugar level, excess body fat around the waist and abnormal cholesterol levels. During times of over nutrition and physical inactivity, changes in the mitochondria, based on increases in storage of glucose as triglycerides in adipose tissue, result from excess supply of electrons via ETF dehydrogenase and succinate dehydrogenase: linking fatty acid oxidation to the ETC. This reaction results in reduction of ubiquinone to ubiquinol, and, in the circumstance of low respiration and high proton motive force, may result in reduction of the electron carrier pools (NADH, flavins, ubiquinone). This detrimental chain of events primes the conditions that favor mitochondrial superoxide production via ETC complex I and complex III. (James, Figure 6) However, increased ROS caused by metabolic syndrome is relatively modest and does not cause cell damage that could be linked with phenotypic changes and risk factors associated with metabolic syndrome. Instead, it is more reasonable to associate ROS in mediated signaling of metabolism that results in changes in cell-metabolism cycles (eg. increased fat storage via inactivation of aconitase). (James)
b) Researchers at the Department of Nutritional Science & Toxicology, University of California, provide a mechanism by which caloric restriction reduces oxidative stress by SIRT3 mediated superoxide dismutase 2 (SOD2) lysine deacetylation. Qiu X et al. proposed this hypothesis based on research done by Palacios OM et al. in which a link between Caloric Restriction (CR) and SIRT3 and AMPK up-regulation. Results showed that “CR diet significantly increased levels of SIRT3 protein in skeletal muscle, compared to the AL control diet” and that “conversely, SIRT3 protein level was significantly decreased following three months of energy-dense, high-fat feeding”. (Palacios, Figure 7) From this stepping stone, Qiu X. et al. that decreases is oxidative stress and damage established by CR required SIRT3 using SIRT3 knockout mice. Because of SOD2’s location: the mitochondria, SOD2 knockout/SIRT3 over-expressing mouse fibroblasts were used to investigate SOD2’s role in the reduction of ROS. Reduction of ROS by SIRT3 over-expressing mouse fibroblasts was blunted in SOD2 knockouts providing evidence of SOD2 as a major downstream component of the CR:oxidative-damage-reduction mechanism. They went on to prove the SOD2 deacetylation mechanism via SIRT3 and the downstream effect of decreases cellular ROS and increases oxidative stress resistance. By measuring deacetylated SOD2 in CR SIRT3 knockout mice vs WT mice, they were able to demonstrate that CR causes SOD2 deacetylation via SIRT3. They also noted that “SIRT3-mediated SOD2 deacetylation and activation was not observed in mice fed Ad libitum”.(Qiu) Overall, these findings demonstrate and extremely thorough and influential mechanism for decreased oxidative damage mediated by Caloric Restriction. (Qiu, Figure 8) “SOD2 activation and increased oxidative stress resistance have been linked to numerous long-lived mouse models” (Qiu) including  a study done by researchers at the  Department of Molecular Gerontology, Tokyo Metropolitan Institute of Gerontology who found that insulin-receptor knockout mice (male and female) “both showed an extended lifespan upon DR” (caloric restriction). (Baba, Figure 9)
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