“How does the cell know when and which proteins to degrade?”
Many researchers are seeking to answer this ultimate question, but even with the extensive research, nobody can really answer clearly. Proteins are continuously synthesized and degraded under regulations. Therefore, protein degradation is as important as protein synthesis. In this paper, ubiquitination, which involves in a protein degradation process, will be discussed along with the Ubiquitin Ligase, a major player in the mechanism.
Ubiquitination is a post-translational modification where ubiquitin, an 8kDa protein that is highly conserved in eukaryotes, is attached to a substrate protein (Lu et al., 2008). Ubiquitin is attached to a targeted protein through a sequential cascade which involves E1 activating, E2 conjugating, and E3 ligase enzymes. This ubiquitin tagging is necessary for many cellular processes, including cell cycle, transcriptional regulation, DNA repair mechanism, apoptosis, protein trafficking, and cell signaling (Miranda M, 2007).
1. Medical Interest
a. Ubiquitin Ligases as Cancer Targets and Biomarkers
The main function of the ubiquitin system was thought to be the role in protein degradation. However, it also plays a role in cellular regulation processes such as cell cycle, DNA damage signaling, and receptor endocytosis (Miranda M, 2007). Defects in this mechanism can lead to various diseases, including neurodegenerative disorders, cancer, viral infection, and inflammation. Hence, it has been a fascinating field for developing cancer targets and biomarkers.
b. Proteasome Inhibitor Drugs Using the Ubiquitin System
Drug targets using the ubiquitin system has been researched and few found drugs have been successful. In 2003, Food and Drug Admini...
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...iquitinated E2 oxyester was incubated and RNF4 was collected on amylose beads.
As in Figure 8 (left), ubiquitin-charged E2 was preferentially bound by RNF4 and free E2 interaction was weak. Also, the free ubiquitin binding was not able to be detected. The model in Figure 8 (right) also suggests the binding of E3 with ubiquitin-charged E2. Through this experiment, RING RNF4 has preference on ubiquitin-charged E2 over other players which allow us to understand the mechanism of ubiquitination better.
5. Conclusion
As a biochemist, I am interested in how the cell works in highly regulated manner. Ubiquitination, the degradation mechanism, has been a fascinating field for many researchers. From this enzyme project, ubiquitination pathway was introduced in step by step. Also, the mechanistic details of RNF4 E3 Ligase were investigated with various experimental data.
... Thus, the loss of BRCA1 can result in defective DNA damage repair, abnormal centrosome duplication, cell-cycle arrest, growth retardation, increased apoptosis, genetic instability and tumorigenesis2. The study of BRCA1 mutation in mice may be used for new therapeutic approaches, although the cause of the mutation in BRCA gene is unclear.
Co-IP is the most commonly used methods to verify protein-protein interactions (Berggård et al., 2007). Antibodies that are specific to the bait complexes are used to capture the bait complexes in a cell lysate shown in Fig. 1. The antibody is immobilized on Protein A/G, which is covalently bound to the agarose beads. Since the antibody is specific to only the bait complex, the antibody will not bind to other proteins found in the cell lysate, and hence, these proteins will be wash off. The antibody-bait compl...
When chemical reactions occur in the body, enzymes are used to speed up the reactions and lower the energy of activation. The rate of reactions is increased due to the complex that is established by the enzyme and the substrate. This complex that forms between the two inhibit movement respective of the two and arranges the reactive groups of the substrates next to each other (1). Enzymes allow chemical equilibrium is able to be maintained throughout the reaction and are not consumed, while accelerating the reaction (2,3). Active sites on the enzymes are where the substrates bind, and the sites are specific to the substrate (2,3). The protein that makes up the enzyme has a particular shape, due to secondary and tertiary structures,
Voet, D., Voet, J., & Pratt, C. (2006). Fundamentals of Biochemistry: Life at the Molecular Level, 2nd edition. Hoboken, NJ: John Wiley & Sons, Inc.
By utilizing the model yeast S. cerevisiae, Ohsumi found a method for identifying the ATG genes. Ohsumi blocked vacuolar degradation using the yeast mutants, then induced autophagy by starving the cells. With the vacuole impaired, the autophagosomes accumulated in the cell, allowing researchers to simply visualize these compartments, which are generally quite difficult to identify. Ultimately, this technique allowed for the discovery of autophagy genes and mechanisms. (4) This concluded the discovery of the first genes necessary for autophagy. The results displayed that autophagy is controlled by a number of proteins and protein complexes which promote a specific stage of the formation of autophagosomes.
Thought to be an oncogene, a gene that has potential in transforming normal cells into tumor cells, p53 was regarded as the most prominent tumor suppressor gene [1]. P53 is a gene which signals apoptosis (programmed cell death) if a cell cannot be repaired due to an extensive amount of damage. As stated in the textbook, p53 regulation occurs by an E3 ubiquitin-protein ligase known as MDM2 [1]. "Controlling the controller" is a statement that describes the molecular interaction where the presence of MDM2 targets the p53 for proteosome via degradation. With three main checkpoints in cell cycle, the literature states p53 functioning from G1 into S phase in a chaotic cell [2]. The normal state of cells is to keep p53 levels low in order to prevent uncontrolled apoptosis and random cell cycle arrest from occurring. In a further note, although p53 promotes apoptosis and cell cycle arrest, cancer may result from p53 unable to recognize the problematic site. In turn, a mutation in p53 may result engaging in new activities. These activities include cellular transformation, tumor metastasis,...
Figure 2: Schematic of Twist1 over activation cascade. The WR domain (N-terminus of Twist1) binds to RELA (NF-kB subunit) inducing the upregulation of
Studies on the VHL gene product, pVHL, have helped clarify cellular responses with changing oxygen availability in mammalian cells, which in turn has helped our understanding on how this product plays a role in heart attacks, cancer, and strokes (Kaelin, William G., 2002). The pVHL protein is primarily located in the cytoplasm, but it has the capability of moving between the cytoplasm and the nucleus. pVHL regulates two types of hypoxia-inducible transcription factors (HIF1 and HIF2) that functions in cellular response to oxygen deficiency (Vaganovs, P., et al., 2013). HIF is a helix-loop-helix transcription factor that consists of two subunits (HIFα and HIFβ) that regulate cellular responses when there is an inadequate supply of oxygen, otherwise referred to as hypoxia (Kim, Jenny J., 2010). HIFβ is continuously produced unlike HIFα, which is highly regulated through ubiquitination by pVHL (Kim, Jenny J., 2010). pVHL also plays an important role in ubiquitination or protein degradation of key proteins within the cell (Kim, Jenny J.,
Interestingly CFTR rarely makes it to the cell surface even in persons expressing wild-type CFTR. The sheer complexity of the protein causes about 70% of CFTR to be moved to a degradation pathway by the endoplasmic quality control system (ERQC) even in healthy individuals
P53 is very significant to apoptosis, DNA damage, and gene stability through complex mechanisms. P53 can respond to DNA damage through DNA repair protein. An example of this would be when DNA damage occurs, p53 gets started and begins the transcription of repair protein XPC and DDB2. The domain p53-XPC-DDB2 can repair the DNA damages(Adimoolam and Ford, 2003). As Well, p53 put a halt to the cell cycle at S, G1, and G2 phase (Agarwal et al., 1995), which can provide time for proteins repair to fix the damaged DNA.
Yurchenko, M., L. M. Shlapatska, and S. P. Sidorenko. (13 Aug, 2012) Figure. Digital image. Experimental Oncology. Morion. Web. 16 Apr. 2105. Retrieved from: http://exp-oncology.com.ua/article/3373/the-multilevel-regulation-of-cd95-signaling-outcome
Consequently, chronic UC patients have an increased risk of developing colorectal cancer (CRC). There are no truly effective treatments for UC at the present. Accordingly, the risk of developing CRC remains high. Thus, the development of novel treatment approaches for this disorder is required to attenuate the inflammatory response, prevent mucosal damage and facilitate mucosal healing (Abimosleh et al, 2012).
The first stage of the process is the unwinding of old strands of the parent DNA molecule. The two strands of the double helix are first separated by enzymes. Then, each strand acts as a template for t...
...off-target effects (Snead, 2013). From previous work, they knew that placing a UNA at the first or second position of one strand of a siRNA would impair the gene-silencing ability of the modified strand. They observed that the 5’ UNA-modified siRNAs was able to block the phosphorylation of 5’-OH synthetic siRNAs, which is a crucial step for siRNAs to be loaded into the RISC to initiate gene silencing. The UNA modification prevented gene silencing of the modified strand, but improved silencing potency of the unmodified strand. This conclusion demonstrates how UNA modifications may be important in a therapeutic context, such as if a virus were to cause a cell to produce siRNas with undesired strand selection properties and poor silencing. The UNA modifications of a synthetic siRNA would be able to decrease its expression by preventing the ignition of the RNAi pathway.
Ubiquitin-proteasome System and a renewed look at the importance of PKA and CaMKII in long term memory development.