A_Study_of_Nonentein__a_Theoretical,_Amateur_Protein_Analysis.

A Study of Nonentein Introduction Since the discovery of the presence of the intracellular protein, nonentein, it has become desirable to learn more about its functions and reactions within the cell; for example, to know more about it which proteins it may interact with, and whether these interactions are both a part of standard cell function and necessary for nonentein to function properly. A more advanced understanding of these aspects will help give an increased insight into the functions of cells and proteins in general and also will give rise to possible future applications for nonentein. Therefore, several procedures are suggested to be undertaken upon nonentein: firstly, Co-immunoprecipitation and Western Blotting in order to discover which proteins nonentein interacts with; secondly, Bimolecular Fluorescence Complementation to determine whether nonentein’s interactions are necessary for normal cell function; finally, protein inhibitors or mutagenesis should be utilised so as to confirm that these interactions are necessary for nonentein’s normal function. I – Determination of the protein-protein interactions of nonentein Immunoprecipitation (IP) is a method often used to analyse protein-protein interactions of suspected complexes. The technique immobilises and removes protein complexes from whole cell extracts. The captured protein can then be identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting. First, the cell is lysed and a whole cell extract is prepared. An antibody specific to the antigen is added to the cell lysis and the mixture is incubated for several hours. During this time, the antibody will interact with the antigen and they will form an immune complex. An agarose gel support – either protein A or protein G – is added to this mixture and – again, over a period of several hours – the immune complex will be bound to the gel. As shown in figure 1, the antibody binds to the gel beads and the antigen (plus its complex) binds to the antibody. Any proteins not bound to the gel support are eluted using an additional buffer and, thus, the antigen is precipitated from the extract. Now, the antigen can be identified using SDS-PAGE and Western Blotting. In using this procedure upon nonentein, however, one is assuming that nonentein behaves like an antibody. This is not known to be true and therefore a Pull-Down Assay may be a better technique to use to discover proteins which react with nonentein. The procedure is similar except nonentein would be used as a bait protein rather than an antibody. Thus, no such assumption is made about nonentein’s character. Also, nonentein would need to be purified before it could be used in the procedure, and tagged with a protein-reactive tag (for example, Sulfo-NHS-LC-Biotin). II – The necessity of nonentein complex for cell function Protein-protein interactions are crucial to many cellular functions, such as DNA replication, transcriptional activation, cell growth and the transduction and translation of intercellular signalling. Do nonentein’s interactions play a part in one of these functions' Immunoprecipitation and Pull-Down Assays are useful in vitro techniques for identifying protein-protein interactions. However, they cannot tell us about the interactions in vivo and the roles they play in the living cell without first disrupting it. Bimolecular Fluorescence Complementation (BiFC) is a technique used to directly visualise protein-protein interactions within living mammalian cells. The process involves fragmenting a fluorescent protein and attaching these fragments to various different proteins, which, when they interact, bring the fluorescent protein together again, as demonstrated in Figure 2. The close proximity of the fragments of the fluorescent protein causes their signals to become considerably stronger than when they were apart on non-interactive proteins and so the locations of the interacting proteins in the cell can be seen and thus their function speculated. There have been many occasions in which BiFC has helped develop ideas about the roles of certain protein interactions within the cell. For example, it was utilised for the identification of several enzyme-substrate complexes. Tom K. Kerppola (2006), in an article titled “Visualization of molecular interactions by fluorescence complementation”, explains: “Determination of the substrate specificities and sites of action of these enzymes in vivo has yielded new hypotheses for their functions. For example, in Saccharomyces cerevisiae, it has been shown that the ubiquitin E3 ligase Grr1 interacts with Hof1, a regulator of cytokinesis within the bud neck during the M phase of the cell cycle.” (Kerppola, 2006) Therefore, by studying nonentein using Bimolecular Fluorescence Complementation, an idea of the role and therefore necessity of its interactions inside the cell can be gained. In a paper entitled “A Human Protein-Protein Interaction Network: A Resource for Annotating the Proteome”, Stelzl et al (2005) describe a procedure through which they succeeded in constructing a protein-protein interaction map in order to better understand the functional organisation of the human proteome. They achieved this by employing yeast two-hybrid interactions followed by co-immunoprecipitation and pull-down assays to map out a network of interactions between proteins. From this, they could gather a better idea of the functional relationships between proteins. Therefore, if a similar technique was to be used to study the interactions of nonentein, it could be deduced whether its reactions are a requirement for cell function. III – The necessity of interaction for nonentein’s function In order to determine whether interaction with other proteins is necessary for nonentein’s normal function, a procedure utilising either protein inhibitors or mutagenesis could be used. The inhibitor(s) or a method of mutagenesis would be directed towards the proteins which interact with nonentein in the cell and render them incapable of function. If the interacting proteins are disabled but nonentein’s function is still carried out, then it can be concluded that nonentein does not need to interact with other proteins in order to function. A protein inhibitor blocks a specific interaction of a protein by being a mimic of the chemical which the target protein “wishes” to interact with. The target protein cannot tell the difference between the inhibitor and its interactor and so the inhibitor is allowed to bind to the reaction site to which the interactor would usually bind. Thus, the target protein cannot interact. Mutagenesis is a process in which the DNA of an organism is altered in order to alter function. It can occur naturally, as observed by Foster and Cairns (1992) when studying the evolution of E. coli; they noted that, when placed in environment that contained lactose as the only food source, the E. coli evolved a lactose tolerance too quickly for the necessary mutation to have occurred randomly and thus concluded it must have been induced by the organism in some way. The concept can be applied in the laboratory by mutation of DNA by use of chemicals or radiation. In our experiments on nonentein, mutagenesis could be used upon a cell to prevent it producing the proteins which interact with nonentein. Therefore, we could observe whether nonentein can function in the absence of interaction. IV – Conclusion Therefore, investigation into the interaction of nonentein with other proteins, and the importance of these interactions for the function of the whole cell and of nonentein itself, would best be investigated by means such as Pull-Down Assays, Bimolecular Fluorescence Complementation and Protein Inhibition or Mutagenesis. Learning more about these aspects will advance our understanding of the role(s) played by nonentein in the proteome and may lead to suggestions of potential future uses for the protein. After which, further study should perhaps be done into the structure of nonentein and into its suitability for any applications for which it may appear appropriate. Word count: 1180 References Foster, P.L. & Cairns, J. (1992) Mechanisms of Directed Mutation. Genetics, Vol 131, 783-789. (http://www.genetics.org/cgi/reprint/131/4/783) Accessed 08-Feb-2010 Harper, J.W. et al. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. 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(http://www.nature.com/nrm/journal/v7/n6/full/nrm1929.html) Accessed 05-Feb-2010 Kerppola, TK (2008). Bimolecular fluorescence complementation: visualization of molecular interactions in living cells. Methods Cell Biol. 2008;85:431-70. (http://www.ncbi.nlm.nih.gov/pubmed/18155474) Accessed 05-Feb-2010 Khalsa, G (date unknown). Western blotting. (http://askabiologist.asu.edu/expstuff/mamajis/index.html) Accessed 03-Feb-2010 Protein (2010), Encyclopaedia Britannica Online. (http://www.britannica.com/EBchecked/topic/479680/protein) Accessed 07-Feb-2010 Protein Interaction: Guide (date unknown), promega.com (http://www.promega.com/guides/protein.interactions_guide/chap4.pdf) Accessed 02-Feb-2010 Protein Protein Interactions (30-Oct-2006), molecularsciences.org. (http://www.molecularsciences.org/proteomics/protein_protein_interactions) Accessed 03-Feb-2010 Reeves, W.H. (1993). Protein-protein interactions and the regulation of cell function. Molecular Biology Reports, Volume 17, Number 3 / April, 1993. (http://www.springerlink.com/content/q67l626043628250/) Accessed 06-Feb-2010 Shiroshima, T, Oka, C & Kawaichi, M (2008). Identification of LRP1B-interacting proteins and inhibition of protein kinase Cα-phosphorylation of LRP1B by association with PICK1. FEBS Letters, Volume 583, Issue 1, Pages 43-48 (5 January 2009). (http://www.febsletters.org/article/S0014-5793(08)00961-7/abstract) Accessed 08-Feb-2010 Stelzl et al. (2005). A Human Protein-Protein Interaction Network: A Resource for Annotating the Proteome. Cell, Volume 122, Issue 6, 957-968, 23 September 2005. (http://www.cell.com/abstract/S0092-8674(05)00866-4) Accessed 06-Feb-2010 ----------------------- Fig.1 IP – mammalian cell-based format. (molecularsciences.org, 2006) Fig. 2 BiFC: a – diagram illustrating the experimental process$N\qŒ‘ªŒ Ž ' f‡§¨9 : < ~ € • ! O V Ì Í Î Ï Ð Ñ Ò ä å ç S Ç Ò ù ü 1JP]xúõñíñíéíåáåáÝÙÕÙÑÍÈÍÄÀ¼À¼¸À¸Ä¸³¥ œ˜Š˜†‚éÀé~é~é~é~h Phbvçh;'jhsU[pic]mHnHu[pic]hO@ of fragmentation and reformation of the fluorescent protein whilst attached to other proteins; b – examples of a cell undergoing BiFC. (Kerppola, 2006)