The Identification Of Phosphoproteins Regulated By PRAK Under Oxidative Stress

Oxygen free. radicals or, more generally, reactive oxygen species (ROS), including·O2,·OH, H2O2, NO-, are products of normal cellular metabolism. ROS is well recognised for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. When the ROS is at low/moderate concentrations, beneficial effects of ROS occur, as for example in defence against infectious agents and in the function of a number of cellular signaling systems. One further beneficial example of ROS at low/moderate concentrations is the induction of a mitogenic response. But if the free radicals is excessive, the potential biological damage is termed oxidative stress. Oxidative stress has been implicated in various pathological conditions involving cardiovascular disease, cancer, neurological disorders, diabetes, ischemia/reperfusion, other diseases and ageing.The excess ROS can damage cellular lipids, proteins, or DNA inhibiting their normal function. Because of this, oxidative stress has been implicated in a number of human diseases as well as in the ageing process. The hydroxyl radical is known to react with all components of the DNA molecule, leading to both the purine and pyrimidine bases damaged and also the deoxyribose backbone. It is known that metal-induced generation of ROS results in an attack not only on DNA, but also on other cellular components involving polyunsaturated fatty acid residues of phospholipids, which are extremely sensitive to oxidation. Mechanisms involved in the oxidation of proteins by ROS were elucidated by studies in which amino acids, simple peptides and proteins were exposed to ionizing radiations under conditions where hydroxyl radicals or a mixture of hydroxyl/superoxide radicals are formed. The side chains of all amino acid residues of proteins, in particular cysteine and methionine residues of proteins are susceptible to oxidation by the action of ROS.Because the body is constantly exposed to a variety of sources of free radicals, reactive oxygen species, in order to maintain the stability of the intracellular redox state, the body produces a series of anti-oxidation mechanism, involve:preventative mechanisms, repair mechanisms, physical defences, and antioxidant defences. Their "steady state" concentrations are determined by the balance between their rates of production and their rates of removal by various antioxidants.The delicate balance between beneficial and harmful effects of free radicals is a very important aspect of living organisms and is achieved by mechanisms called "redox regulation. The process of "redox regulation" protects living organisms from various oxidative stresses and maintains "redox homeostasis" by controlling the redox status in vivo. A number of studies reported that the serine/threonine kinases of the MAPK family can be regulated by oxidants. There are four known MAPK families:extracellular-regulated (ERKs), c-jun-NH2-terminal kinase (JNKs), p38 MAPK and the big MAPK-1 (BMK-1), of which serine/thereonine kinases are important in the process of carcinogenesis including cell proliferation, differentiation and apoptosis. Products of NOX1 activity, superoxide, hydrogen peroxide can activate the MAPK cascade at the level of MEK and ERK1/2. The experimental studies on the up-regulation of MAPKs by H2O2 treatment have shown that the activation of each signaling pathway is type-and stimulus-specific. For example, it has been reported that endogenous H2O2 production by the respiratory burst induces ERK but not p38 kinase activity. Conversely, exogenous H2O2 activates p38 kinase, but not ERK in rat alvedor macrophages. The ERK pathway has most commonly been associated with the regulation of cell proliferation. The balance between ERK and JNK activation is a key factor for cell survival since both a decrease in ERK and an increase in JNK are required for the induction of apoptosis.PRAK (p38-regulated and-activated kinase), also known as MK5, first discovered in 1998, is an important serine/threonine protein kinase regulated by p38 in the downstream of the MAPK signal transduction pathway. PRAK and other MKs all have nuclear localization and nuclear export signal, but what in particular is that the two localization sequences of PRAK overlap, suggesting its localization in the cell has a special regulatory mechanism and also it has some special function compared with other MKs. Previous experiment showed that PRAK plays an important role in the cell during oxidative stress. When stimulated by exogenous hydrogen peroxide the death rate of PRAK-/- cells was significantly higher than PRAK+/+ cells. But the specific mechanism is still not clear. Therefore, this study was designed to reveal the protein phosphorylated by PRAK in the process of cellular oxidative stress.According to traditional research methods which based on the existing work of PRAK, some molecules interacting with PRAK in the process of oxidative stress response could be derived. And with a detailed study we can find one or several important molecules. But this traditional research strategy is difficult to achieve. First, there is less research to provide reliable and effective for the analysis of PRAK currently; Second, this study, focusing on one or several proteins,is difficult to explain the whole problem. Only when this "point" of the results are accumulating enough, the system's problems will become gradually clear up, but this process generally takes a long time.The proteome is highly dynamic, even if the same cells in different physiological or pathological conditions, the expression of the highly dynamic proteome is different, and this difference protein often just a sign of some disease. Using comparative proteomics research techniques, we can observe differences of the protein expression profile in normal and disease cells (tissue), screen and explore potential functions proteins and markers for early diagnosis, intervention and treatment of disease.As a large-scale, high throughput, high sensitivity method, proteomics can effectively analyze the overall intracellular proteins. However, the protein composition of cells or tissues is so extremely complex that the development of proteomics is restricted and driven by the technology. The study of Proteomics depends largely on the level of their skill level.Fluorescence differential gel electrophoresis (DIGE) is a method which labels protein samples with different fluorescent dyes before 2-D electrophoresis, and then to three different protein samples are separated up at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the biological differences really, while avoid influence of systematic errors. Since the most obvious advantage of DIGE system is integrating the advantages of both CyDye multiple labeling method and DeCyder difference 2-D analysis software. DeCyder software takes the advantage of the spots co-detecting algorithm, which can automatically detected fluorescence images, eliminate background, quantify, normalize and match spots in gel, thus, systematic errors caused by different operators can be eliminated.Protein phosphorylation is the most common as well as the most important type of protein post-translational modification. The activity of many proteins is regulated by the post-translational modification, particularly PRAK as the important serine/threonine protein kinase in MAPK signal transduction pathway, its regulation of the downstream proteins be achieved by phosphorylation. So looking for the proteins regulated by PRAK at the process of oxidative stress is helpful for not only Comprehensive and in-depth understanding of PRAK, but also Searching for effective endogenous antioxidant pathway.Based on such considerations, we design a differential proteomics experiments by stimulated normal and PRAK-/- MEF cells 45min with NaAsO2. Then cytoplasm phosphoproteins were extracted by the phosphate metal affinity chromatography resin (PMAC). Finally, the differential expression of protein profile of four groups has been established by using DIGE technology. Through analysis by use of DeCyder difference 2-D analysis software, 32 differential protein spots were found. The numbers of up-regulated proteins is 18 in the normal MEF cells after NaAsO2 stimulated and 11 in the PRAK-/- cells; while the numbers of down-regulated proteins is 2 in the normal MEF cells after stimulated and 1 in the PRAK-/ cells. At last, a total of 16 proteins have been identified among these differential protein spots by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/TOF). 13 of them are confirmed to be phosphorylated.We predicted the subcellular localization of the differential proteins and made a result that, most proteins locate in cytoplasm and nucleur. After subcellular localization analysis, the protein functional analysis has been done. Through analyzing protein domain and motif database, the identified proteins roughly included Membrane protein, oxidoreductase, Ion channel protein, Cytoskeletal proteins, Cytokines. However, we also predicted protein interactions using the "String" protein interaction databases. We found that Interacting proteins were foundless.Based on the researches above, we have some conclusion:1. Cytoplasm phosphoproteins were enriched through the phosphate metal affinity chromatography resin (PMAC). Subsequently, the phosphoproteins of control and stimulated (NaAsO2 45min) normal and PRAK-/- MEF cells were separated by DIGE.32 differential protein spots with statistical significance were detected.2. The significant differential protein spots were analyzed by mass spectrometry and 16 proteins were identified after deleting keratin and duplication proteins.13 of them have had confirmed to be phosphorylated.3. We predicted the subcellular localization of the differential proteins and made a result that most proteins locate in cytoplasm and nucleur.4. Through analyzing protein domain and motif database, the identified proteins roughly included membrane protein, oxidoreductase, Ion channel protein, Cytoskeletal proteins, Cytokines.5. We also predicted protein interactions using the "String" protein interaction databases. The result that interacting proteins were foundless, shows the process of oxidative stress regulated by PRAK involved many aspects of cell function, rather than a complex of proteins share the same function.6. Analysis the phosphorylation level of these 16 proteins in different cells and found that in the process of oxidative stress, some proteins directly regulated by PRAK, and a small amount of proteins may be regulated through a compensatory pathway when there is no PRAK expressed and some proteins may be regulated through specific protein which phosphorylated by PRAK.