Evaluating P2x7 Receptor & Cdc25 Proteins 14

P2X7 also known as “purinoceptor” is a famous gene related protein that is found in humans beings and is encoded and related to the P2RX7 gene family. The root of this gene is related to the family of purinoceptors for ATP. In this research paper, we will start by explaining the functional properties of the P2X7 receptor and will study how overexpressing of P2X7 protein receptor is done in HEK293 cells. Moreover, we will discuss on regulation of Cdc25 proteins at the G2/M checkpoints and the activity related to the p38 kinase when exposed to UV irradiation.

Lastly we will perform and describe the experiment done on two different cells say A and B and will find which one is sensitive and which one is resistant.

P2X receptors are nonselective cationic channels gated by extracellular ATP and are communicated by an assortment of mammalian cell types including neurons, glia, epithelial cells, and smooth muscle cells. Sufficient proof shows that particular subsets of P2X receptors are engaged with preparing torment signals at fringe and spinal dimension. M.T. & Piwnica-Worms, H. (1998)

Specifically, the expression of the P2X7 receptor is used mostly in non-peptidergic small-diameter dorsal root ganglion (DRG) neurons, which are connected with P2X3-dependent nociception that transmits the signals of pain. There are many studies done that shows that overexpressing of P2X7 receptors results in a behavioral effects like hyperalgesia and allodynia.

There are many further types or say subsets of the P2X receptor protein, from which the type-C receptor has found to contain the most of the gene and homology sequence, but still the purification and overexpression of P2X7 receptors with respect to mammalian cells remain poorly understood Pines, J. (1999). However, the studies and knowledge regarding the most types of P2X receptor remains limited but the main function of these receptor proteins has found to be the they up-regulate and down-regulate depending on the state of their phosphorylation. Peters, J.M. (2002)

Culturing cells and reagents: There are many studies done that have revealed the proven results of overexpressing P2X7 receptors in HEK-293T cells, moving forward in this report we will discuss how this method works and what results have been generated by the using this method

So in this method the set of HEK-293T cells are regularly monitored and regularly cultures of cells are made in DNEM medium plus 10% FCS. Now we will perform Cell transfection of plasmids pCDNA3-P2X3, pCDNA3-P2X3-Y393A encoding rat WT or mutated P2X7 receptors with calcium phosphate Raleigh, J.M. & O’Connell, M.J. (2000). In this method the Control method used is “pEGFP plasmid” to control. Furthermore HEK-293T Cells were analyzed 24, 48, or 72 h after transfection. Elledge, S.J. (1997)

According to the above mentioned procedure it is found that P2X7R expression allows proliferation of HEK293 cells, when deprived and combined with serum and glucose. It was revealed that P2X7R-transfected HEK293 cells have a higher lactate outcome, overexpress several of the key glycolytic enzymes and the ubiquitous glucose transporter Glut1, have larger glycogen depots and show an increased level of phosphorylated Akt/PKB (ph-Akt/PKB) and hypoxia-inducible factor 1? (HIF-1?).

In low glucose, expression of glycolytic enzymes is strongly up-regulated in HEK293-P2X7, much less in wild-type or mock-transfected HEK293 (HEK293-mock) cells Beach, D. & Roussel, M.F. (1999). These results show that P2X7R expression allows better adaptability to unfavorable ambient conditions via up-regulation of glycolytic enzymes and by increasing intracellular glycogen stores, and may help to better understand cancer cell energy metabolism and tumor progression and dissemination. The results of the study can be found in figure 1.

A crucial protein/enzyme used for controlling glycolysis is G3PDH, its primary role is that it is responsible for the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. The G3PDH gene is mostly regulated for the identification or monitoring of glycolytic metabolism mostly in the growing cancer cells in a host. The other most used control proteins are PFK, PKM2 and PDHK1 Shiloh, Y. (2001). Multiple studies revealed that G3PDH levels are over three-fold higher in HEK293-P2X7 versus HEK293-mock cells due to high high glucose.

In cases with low glucose, G3PDH expression increases further in both cell types, but to a much larger extent in HEK293-P2X7 than in HEK293-mock cells.While assessing other protein control PFK the studies shows that expression of PFK is higher in HEK293-P2X7 than in HEK293-mock cells, albeit level of expression is not significantly changed by glucose depletion. And in low glucose, level of expression of PKM2 is enhanced in HEK293-P2X7 compared with HEK293-mock cells. Under these conditions, PDHK1 is also overexpressed in HEK293-P2X7 cells see figure 2.

As according to the similarities found in mammalian Cdc25 homologues of both mechanistic and structural, the biological difference between different mammalian cdc25 proteins are mostly identified when they are activated, subcellular localized and also can be reorganized by the levels of phosphates present. The regulation of cdc25 phosphates is carried out on multiple levels while most cdc25 proteins have a constant cell cycle and identification process. But during the process of regulating both types of proteins i.e. Cdc25A and Cdc25B fluctuate from time to time. The mechanisms that take part in regulation and identification of different cdc25 proteins are summarized in below table 1.

We can determine the p38 kinase activity in damage DNA or cells by infusing Cdc25 phosphatases into the G/2 M checkpoints. While regulating and maintaining the Cdc25 proteins at the G2/M checkpoint these checkpoints can be marked as Chk1, Chk2/Rad53, but the names of proteins and mechanisms responsible for activation and regulation are still not clear and defined. However, according to many studies done in this report we report that p38 kinase has a specific responsibility when it comes to the activation of the G2 delay after been exposed to UV radiations.

Studies shows that p38 kinase restricts the fast activation of the G2/M checkpoints in both mammalian and humans cases when exposed in ultra violet radiations. Recent progressions in this area has revealed a new mechanism/method for regulating cdc25c proteins , according to that procedure through ubiquitin-dependent, meditated degradation there is a high chance that G2 arrest might occur. And studies have proved that regulation of cdc25 proteins can cause and abrogation in p38 kinase cells when exposed to UV radiation at the G2/M checkpoints. J.M. & O’Connell, M.J. (2000)

While infusing Cdc25 phosphatases into the G/2 M checkpoints different types of proteins like cdc25c and cdc25b are get affected and targeted by damaged DNA check points. When all these proteins are exposed under the UV radiation the p38 kinase cells abrogate and all cells get activated but under some specific circumstances some sets of proteins like cdc25A can get in-activated while rest of the proteins are activated this can cause a block in the process and stop the cells cycle that can lead to G2 arrest. As cdc25A is very un-stable protein and the degradation of this protein can go very fast in the G2/M or damaged protein checkpoints Takizawa, C.G. & Morgan, D.O. (2000).

Accelerated Cdc25A degradation, as a result of phosphorylation by the Chk1 and/or Chk2 protein kinases, results in sustained inhibitory phosphorylation of Cdk2, leading to G1 arrest and a block in S-phase entry . Likewise, a failure to dephosphorylate and activate Cdk2 results in an S-phase delay, as it prevents the loading of the essential replication factor Cdc45 and, in turn, recruitment of DNA polymerase to replication origins. In human cells, the integrity of the ATM/Chk2/Cdc25A/Cdk2 pathway was found to be required to prevent radioresistant DNA synthesis . In the presence of double-stranded breaks (DSBs) caused by ionizing radiation, cells activate the ATM protein kinase, which has a broad range of target proteins, including Chk2. The activated Chk2 then phosphorylates Cdc25A on Ser 123, targeting the phosphatase for ubiquitin-dependent proteasomal degradation. Takemasa, I. et al. (2000)

There are many different ways that can help provide an insight which cell is sensitive or resistant. In our given case for this paper, we have 2 tubes say A and B containing cells of different types and we don’t know which one of the tube contains the sensitive group of cells and which tube contains the resistant group of cells Taniguchi, E. & Nishida, E. (2002). For this purpose we will have to conduct an experiment that will provide us the information we need to know.

There are different methods and experiments many studies have used in this paper I will be using the technique of identifying the cells by dividing the tubes into two types one is we assume contains necrotic cells and the other contains apoptosis cells. The whole idea of experiment is to distinguish the cells by evaluating the type of cell death inhibited. Zhou, B.B. & Elledge, S.J. (2000)

Dividing the cells: First we start off by assuming one tube contains necrotic cells and the other contains apoptosis cells. Apoptosis is a process of cell death occurring due to natural reasons or it is also widely known as controlled cell death. There are no toxic levels present in this type of cell death. So basically what it means is that the cells that shows the characteristics of a natural cell death and has no toxic level means that are apoptosis cells and are hence, sensitive.

And on the other hand, Necrotic cells are the ones that die because of any toxic levels and or because of any disease or any injury suffered by the host. So the cells that show high toxic levels will be necrotic cells, that are mostly resistant. So this is the basic concept by which we can distinguish the cells from one another just by examining the way of death suffered by cells. The procedure of finding that wither a cell is apoptosis or necrotic is explained below:

TUNEL and DNA damage Assay: Out of many different procedures to identifying cell types TUNEL and DNA damage is widely used and famous method. TUNEL known as (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) works by identifying the broken down DNA pieces and identifies the color of the cells which seems to be brownish in apoptosis cells.

Caspase detection assays: We can use this method to detect the necrosis cell types that contains toxins levels and the death cycle inhibits non-natural death reasons including different diseases, attacks and injuries. In this method a small segment of cells are placed under the microscope and examined for inflammatory characteristics and change of color which in the necrosis cells case is turned into black, due to toxins levels present in it.

References

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