G-protein-coupled receptors (GPCR) consists of the largest family of drug targets. Many signaling systems depend on GPCR to convert external and internal stimuli to intracellular responses. They consist of a single polypeptide that is folded into a globular shape and embedded in a cell’s plasma membrane. They are also called seven-transmembrane receptors because of the presence of seven segments that run across the entire length of the cell membrane. GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule binds to a GPCR, it causes a conformational change in the GPCR. This change then triggers the interaction between the GPCR and a nearby G protein. Whenever a G protein is active, both its GTP-bound alpha subunit and its beta-gamma dimer can relay messages in the cell by interacting with other membrane proteins involved in signal transduction. Specific targets for activated G proteins include various enzymes that produce second messengers, as well as certain ion channels that allow ions to act as second messengers. Some G proteins stimulate the activity of these targets, whereas others are inhibitory. As is the case with other membrane proteins, GPCRs are subjected to a stringent quality control mechanism at the endoplasmic reticulum, which ensures that only correctly folded proteins enter the secretory pathway. Because of this quality control system, point mutations resulting in protein sequence variations may result in the production of misfolded and disease-causing proteins that are unable to reach their functional destinations in the cell.
One mechanism through which the mutation could cause a lack of proper targeting is protein overexpression. Overexpression can simply reduce the steady-state levels of other proteins, by affecting their transcription, translation, or their rate of degradation. They can also activate new pathways through neomorphic effects. For instance, overexpression of cytoplasmic protein could lead novel phenotype due to the accumulation of sub-population in the nucleus.
The main reason for doing experiment shown in figure 3 was to characterize the larger role of the cargo binding domain of Sec24p protein sorting. The experiment was done by the bay number of cargo molecules that in the vesicles and which are produced with Sec24L616W mutant. From this experience they learnt that the fidelity of cargo sorting was maintained when vesicles were generated with the mutant Sec24p. They also discovered that the ER residents Sec61p and Kar2p were not found in the vesicle fraction but A particular group of cargo molecules was missing from the vesicles generated with mutant Sec24p with respect to wild-type vesicles. Some cargo molecules such as pro–factor and a chitin synthase and Chs3p were packaged efficiently with mutant Sec24p. The Erv41/Erv46p complex, which employ dihydrophobic and aromatic export signals were also packaged normally. The packaging of the general amino acid permease Gap1p that employs a diacidic motif that is needed for efficient ER export was not affected by the Sec24p sorting mutation. The packaging of Hip1p and Can1p was unaffected by mutation of the cargo binding domain. The intermediately affected proteins included the SNAREs (Bos1p and Sec22p) and the p24 proteins Emp24p, Erp1p and Erp2p, which contain uncharacterized sorting signals. These proteins all share a pair of hydrophobic residues at their extreme C termini involved in ER export. The most severely affected proteins included Bet1p and a Gap1p/Sys1p chimera, which depends entirely on the Sys1p diacidic motif for uptake into COPII vesicles.
If the amino acid signal DID in the protein Gap1p was mutated to random amino acids (LxxLE) the following results are expected. The Gap1p present will require diacidic motifs to bind COPII vesicles that are needed to transport complex cargo proteins to Golgi bodies.
The two possible results that might occur if the amino acid signal LxxLE in Bet1p was mutated to the amino acids DID are shown in Figures (a) and (b) below. This happens because Bet1p receives diacidic motifs that are necessary to bind COPII vesicles the packaging of cargo proteins.
During the experiment shown in Figure 5C, bacterially expressed GST-CDC25C or GST was incubated with recombinant AMPK or ERK in an in vitro phosphorylation assay. Proteins were resolved by SDS-PAGE and immunoblotting for the indicated antibodies.
The bacterial expression vectors for GST-tagged CDC25 fragments were generated by PCR amplification of the encoding human CDC25C cDNA, followed by sub-cloning of the cDNA into pGEX-4T3 vector. Activated recombinant ERK (p42 MAPK) was prepared by incubating MBP-MAPK with constitutively activated MBP-MKK1 (115). GST- and MBP tagged recombinant proteins were expressed in bacteria and purified as described previously. The kinase reactions were performed in 10µl of reaction mixture (1µl of 10 × kinase buffer and 0.25 mMATP), AMPK (1 unit), and 1µg of the indicated substrates for 30 min at 30 °C. The reaction mixtures were boiled in SDS sample buffer and subjected to SDS-PAGE analysis and a Western blot assay.
This experiment was done to generate recombinant CDC25C fused to glutathione S-transferase (GSTCDC25C) and a non-related GST-tagged protein and phosphorylated these proteins with recombinant AMPK or extracellular signal-regulated kinase 1 (ERK1) or PBS control. According to the experiment it was learned that ERK readily phosphorylated CDC25C at threonine 48, as we reported previously (49). However, only the AMPK-mediated phosphorylation site in CDC25C was recognized by the phospho-AMPK substrate motif antibody or the pSer-216 antibody.
Another control experiment would involve LKB1 immuno precipitates which are used to phosphorylate MBP–AMPK in vitro, and then MBP–AMPK removed and tested for its ability to transphosphorylate the SAMS peptide in the presence of ATP. Samples without LKB1, without SAMS peptide, or without MBP–AMPK all gave similar levels of background.
The active form of LKB1 alone is incapable of detectably phosphorylating the SAMS peptide. This is because LKB1 phosphorylation of MBP–AMPK activates its kinase activity toward a peptide substrate.
Cyclin-dependent kinases (CDKs) play a critical role in the cell cycle. These are a family of sugar kinases. They not only regulate the cell cycle but also are involved in the mRNA processing, regulation transcription and differentiation of nerve cells. CDKs are involved in the addition of negative phosphate group through the process called phosphorylation. Through this process, CDKs bind to the cell signaling it to move the next phase in the cell cycle. They depend on cyclins. Cyclins are helpful because they bind to CDK thereby activating them to phosphorylate and other molecules.
Cyclin proteins are involved in the process of synthesis and degradation in cell division. During the synthesis process, they activate the protein and bind to CDKs thereby forming cyclin-CDK complex. This new product formed make it possible for the cell to move to the next step of the cell cycle. When the cyclin degrade they deactivate the CDK enzyme thereby exiting from the specific phase.
The two classes of cyclins involved in this process are G1 cyclin and mitotic cyclin. G1 cyclins bind to the CDK and they make the cell to exit from G1 and enter into S phase. During the degradation, G1 cycle deactivate CDK making the cell to exit into S phase. Mitotic cyclin gather during G2 and when they attain high concentration they bind CDK in G2 leading to the formation of mitosis promoting factor, otherwise known as MPF. MPF allow the cell to enter the mitosis process. During degradation process, MPF becomes inactive and the cell leaves mitosis to re-enter G1.
An example of positive control is the regulation of growth factors period on the other hand an example of a negative control is the DNA damage.
Apoptosis is there any nation of cells from the body without causing or leaving harmful substances in the body. During cell cycle regulation old cells, unhealthy cells and unnecessary self are removed from the body. Apoptosis and cell cycle regulation are related because apoptosis help in maintaining the health status of the body.