IntroductionThe H. Freeman, 2008, p.935.). RAS proteins are,

IntroductionThe Literature I am reviewing for my Library Project relates to the structure of the isoprenylcysteine carboxyl methyltransferase (ICMT) enzyme and how implementing different mechanisms of inhibition will cause an irreversible cascade which ultimately results in the enzyme’s inactivation. Inhibition of this enzyme is particularly important in the RAS signal transduction pathway as ICMT is a necessity for the RAS pathways because it is responsible for the maturation of the RAS GTPases (Guanine triphosphate) for them to function in gene regulation and cell proliferation. The link between this enzyme and RAS G proteins is oncogenesis. Oncogenesis is the termed coined for the development of cancer within the cells of the body (Lodish, H. (2008). Molecular Cell Biology. 6th ed. W. H. Freeman, 2008, p.935.).  RAS proteins are, in fact, oncogenes/proto-oncogenes which means that they are proteins encoded by DNA that are at a significantly higher risk of becoming cancerous cells. Due to RAS GTPases having a direct link to gene regulation, cell proliferation and the production of second messenger systems, they are an ideal target to aid in cancer metastasis. If mutated, the cancer retains the ability to keep the RAS proteins in their active Guanine nucleotide Exchange Factor (GEF) bound state. Once the RAS is constantly active the cancer can utilise the open pathway to manipulate cancer cell proliferation, promoting the growth of cancerous cells and the spread of the cancer through the blood stream to other parts of the body (Lodish, H. (2008). Molecular Cell Biology. 6th ed. W. H. Freeman, 2008, pp.936-937). Ergo by inhibiting the intramembranous enzyme ICMT, it will not mature the RAS proteins into its active form and as a result cancer cannot manipulate the RAS pathway to induce metastasis. The article that I am reviewing investigates the crystalline structure of the ICMT enzyme in the endoplasmic reticulum and by utilising different methods, how the enzyme can be destabilised and thus aiding in the prevention of RAS- driven cancers (Diver, M., Pedi, L., Koide, A., Koide, S. and Long, S. (2018). Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT. Nature, 553(7689), pp.526-529.).Structure of ICMTThe structure of the ICMT enzyme in the intramembrane of the endoplasmic reticulum is essential to the article as the purpose of the paper is to deliver a detailed image of the structure which can be further investigated to fully determine how exactly the enzyme may be inhibited. This paper is a follow on from another article produced by MM. Diver et al in 2014 where the structure of the enzyme was manipulated to induce inhibition by mutations of essential amino acid residues within the active site and cofactor pocket (Diver, M. and Long, S. (2014). Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites. Journal of Biological Chemistry, 289(38), pp.26007-26020.). The enzyme’s structure is essentially a class four integral protein with eight alpha-helical domains called M1 – M8 that rests in a typical phospholipid bilayer. The structure retains a short cytosolic N-Terminus and a long cytosolic C-Terminus. The N and C terminals refer to the backbone structure of a protein where the N-Terminus stands for an NH3 region and the C-Terminus stands for a COOH region, in other words an amino group and a carboxylic acid. The alpha-helices themselves are interconnected by short proline residues in the luminal leaflet, which is typical for large membranous proteins for stabilisation. In addition, the structure contains multiple stabilising amino acid residues, that form hydrogen bonds to not only ensure total security to the overall structure but specifically to the active site where the environment is rich in aromatic amino acid residues (Yang, J. et al (2011). Mechanism of Isoprenylcysteine Carboxyl Methylation from the Crystal Structure of the Integral Membrane Methyltransferase ICMT. Molecular Cell, 44(6), pp.997-1004). The helical domains M1 and M2 share non – covalent interaction with the active site in conjunction with the enzyme’s cofactor further attributing to powerful stabilising forces for this key enzyme. A cofactor or coenzyme is an essential organic molecule or inorganic ion that is responsible for allowing the powerful function of the ICMT enzyme (McMurry, J. (2018). Organic Chemistry. 9th ed. Cengage Learning, p.898.).  The coenzyme for ICMT is known as S-adenosyl-Lhomocysteine and can only be oriented in the cofactor pocket when it is released and S-adenosyl methionine is bound instead which is mediated by hinge point residues, allowing for flexibility in the enzyme.The cofactor pocket is structurally secure by interactions with the M6 and capping protein on the C-terminus. It is a requirement for the enzyme to facilitate the activation of the enzyme, therefore it will have stabilising regions in which the active site will be associated with. The active site itself has links to M1 and M2 and consists of many hydrophobic amino acids and polar amino acids.The reason ICMT would be found in the endoplasmic reticulum is due to RAS having to be sent to the plasma membrane in a secretory vesicle. RAS must be matured by ICMT prior shipping to the cell membrane.Figure 1a: ICMT two-dimensional structure.This figure is a two-dimensional rendition of the ICMT structure. It outlines the enzymes general structure,Indicating the positions of the helical domains, the terminals, the stabilising residues and proline motifs. Italso conveys the position of the active site and the substrate binding regions and its associated stabilising residues. Source: Diver, M. and Long, S. (2014). Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites. Journal of Biological Chemistry, 289(38), pp.26007-26020.Figure 1b: Hydrogen bondsThis figure outlines the hydrogen bonds found between the amino acid residues of the ICMT structure. The Dashed lines denote hydrogen bonds.Source: (Yang, J. et al (2011). Mechanism of Isoprenylcysteine Carboxyl Methylation from the Crystal Structure of the Integral Membrane Methyltransferase ICMT. Molecular Cell, 44(6), pp.997-1004). Figure 1c: The active site The active site of the enzyme outlined here in three – dimensional form shows the residues that stabilise the active site. Seen here isThe S- Adenosyl – L – homocysteine AdoHcy which is an amino acid derivative essential to the enzyme.Here the dashed lines also denote the presence of Hydrogen bonds.Source: (Yang, J. et al (2011). Mechanism of Isoprenylcysteine Carboxyl Methylation from the Crystal Structure of the Integral Membrane Methyltransferase ICMT. Molecular Cell, 44(6), pp.997-1004). Protein Substrates and RAS MaturationICMT’s enzymic functions are utilised for the maturation of the RAS G proteins. Not only is ICMT responsible for maturing RAS G proteins but it is responsible for more than two hundred other intercellular proteins. However, ICMT acts upon specific proteins known as CAAX proteins. CAAX proteins must undertake three cellular process but only one is specific to ICMT. ICMT is essential to ensure complete maturation of the GTPase, steps include: polyisoprenylation, proteolysis and carboxyl methylation (Gao L, Liao J, Yang GY. CAAX-box protein, prenylation process and carcinogenesis. Am J Transl Res. 2009;1:312–PMC free article PubMed). The first two steps are vital as they are key to additions of prenyl groups and removal of the -AAX terminal of the CAAX protein but, the final step is the most pivotal step as it is in this step where ICMT is solely in control of the methylation of its prenylcysteine substrates, thus maturing molecules like RAS GTPases. Once matured the Ras GTPases are essential to many signal transduction pathways especially Receptor Tyrosine Kinase pathways (RTK) (Gao L, Liao J, Yang GY. CAAX-box protein, prenylation process and carcinogenesis. Am J Transl Res. 2009;1:312–PMC free article PubMed). In this pathway the RAS proteins are activated downstream of the transmembrane receptor. Once a molecule such as insulin Growth Factor (IGF) or Epidermal Growth Factor (EGF) binds to the RTK, the receptor dimerises and auto-phosphorylates. The phosphate molecules on the cytosolic leaflet of the receptor binds the SH2 and the SH3 (GRB2) proteins which in turn bind the Sos protein. The Sos protein facilitates the conversion of RAS -GAP to RAS -GEF. Once in the active GEF bound state the RAS protein can cause an autophosphorylation cascade via multiple other similar proteins until specific proteins bind to DNA to regulate gene expression. Other proteins include: RAF, ERK, MAP kinases.  RAS proteins are explicitly limited to the plasma membrane of the cell and do not have locomotion around the cytosol (Wright, L. and Philips, M. (2006). Thematic review series: Lipid Posttranslational ModificationsCAAX modification and membrane targeting of Ras. Journal of Lipid Research, 47(5), pp.883-891.).The question that must be asked here is why is this important in the struggle against cancer? If a RAS GTPase were to become permanently active by a mutation then gene regulation would be constantly active, because of this cancer can manipulate the RAS signal transduction pathway to induce cancer cell proliferation and further metastasis within the body. This can make cancers more aggressive and more difficult to cure. Thus, it is quite feasible to determine that RAS G Proteins are oncogenic.Figure 2a: RAS Structure This diagram outlines the structure of a matured RAS G protein in both its GEF and GAP forms (active and inactive structures).The diagram was obtained via the same method as the ICMT figure which is known as X-Ray crystallography. It is a technique used to figure out atomic and molecular structure of a crystal Source: Lodish, H. (2008). Molecular Cell Biology. 6th ed. W. H. Freeman, 2008, p.542.Figure 2b: RAS Pathways Here the RAS signal transduction pathway is evidently projecting from the centre conveying its wide reach. Notice must be paid to MEKK, JNK, RAF and ERK as these are the typical proteins manipulated in the cancers rigid progression.Source: Malumbres, M. and Barbacid, M. (2003). Timeline: RAS oncogenes: the first 30 years. Nature Reviews Cancer, 3(6), pp.459-465. Figure 2c: Cancer FormationOutlined in this figure is RAS proteins become cancer cells. Being a proto-oncogene, RAS is readily manipulated and mutated. In addition to this, the “guardian of the Genome” p53 is a tumour protein that is responsible foor surpressing tumours (Solomon, H., Madar, S. and Rotter, V. (2011). Mutant p53 gain of function is interwoven into the hallmarks of cancer. The Journal of Pathology, 225(4), pp.475-478.). If these two cells are mutated then cancer is an inevitability.Source: Luo, J. and Elledge, S. (2008). Deconstructing   oncogenesis. Nature, 453(7198), pp.995-996.ICMT and OncogenesisIsoprenylcysteine carboxyl methyltransferase is an essential part to almost all metabolic pathways connected to RAS signal transduction pathways. With RAS being interconnected to many ionotropic and metabotropic cell surface receptors it is a contact point for other receptors that don’t have monomeric G protein signallers. As aforementioned this makes the RAS protein a target for mutation by cancer as by doing so it gives cancer the ability to become truculent. It gives cancers an advantage against host defences and clinical therapies because oncogenesis within these specialised proteins gives access to mobility laterally in and out of other pathways but to also continue mutating downstream, avoiding the need to be reliant upon one protein. The ability to be mutated stems from the genome. The genome consists of approximately twenty thousand to twenty-five thousand genes, which is a massive number of genes that require strict regulation. Out of that number there are approximately twenty-one thousand proteins that are or can be expressed by a human. RAS has some isoforms, but they do not add up to a significant amount within the proteome. Therefore, mutations and deregulations are quite understandable in the context of the big picture and it becomes increasingly evident as to why RAS proteins are the target of aggressive cancers (Wang, T., et al. (2017). Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras. Cell, 168(5), pp.890-903.e15.).Figure 3: RAS CancerThis Figure was extracted from an online site dedicated to cancer research. This image shows us a benign RAS mutation that is pre-operation as there is a high risk of the mutation becoming malignant.Source: Guttler, D. (2018). RAS Mutation in “benign”( NIFT-P)Non invasive follicular tumor with papillary cell features – Thyroid Center of Santa Monica. online Thyroidnosurgery.com. Available at: http://thyroidnosurgery.com/thyroid-cancer/ras-mutation-in-benign-nift-pnon-invasive-follicular-tumor-with-papillary-cell-features/ Accessed 30 Jan. 2018.ICMT and Progeria Mutations in the substrates of the ICMT enzyme are not solely linked to GTPases. Prelamin A is another typical protein that must undergo the three-step process: polyisoprenylation, proteolysis and carboxyl methylation (Gao L, Liao J, Yang GY. CAAX-box protein, prenylation process and carcinogenesis. Am J Transl Res. 2009;1:312–PMC free article PubMed) in order for it to mature to become active. The substrates linked to ICMT all converge on the point of being disease related and prelamin A is no different. Prelamin A is a nuclear associated, class five intermediate filament. It is a fibrous protein that provides essential support to the regulation of transcription and because of its pivotal role in the cell prelamin A is associated with disease. This disease is Progeria. Progeroid diseases include most famously, Hutchinson-Gilford progeria syndrome and restrictive dermopathy and these both arise because of prelamin A’s association with ICMT. Progeria is a fatal condition that is distinguished by rapid ageing in children. Most children do not survive to their teenage years and most often die of heart disease (Ibrahim, M., et al. (2013). Targeting Isoprenylcysteine Methylation Ameliorates Disease in a Mouse Model of Progeria. Science, 340(6138), pp.1330-1333.).Figure 4: Mouse Model of ProgeriaThis is a mouse model of the effects of Progeria. As evident on the image the mouse on the left looks considerably ages, deformed. There is a clear issue with its joints as well as improper growth of the body and hair.Source: Johnson, T. (2013). Rapid Aging Rescue?. Science, 340(6138), pp.1299-1300.ICMT INHIBITION  Inhibition of ICMT has extreme possibilities for utilisation against aggressive diseases such as those previously mentioned. ICMT has multiple modes of inhibition: structural mutations, Monobody insertion and nucleophilic attack. By inhibiting the linking agent between Ras-cancers and prelamin A-progeria, ICMT will no longer be able to mature these proto-oncogenes which might be useful for deciding novel clinical treatments. Monobody: Monobodies are synthetic binding proteins that are created using randomised fibronectin protein domains. These are molecules that can be substituted for antibodies in a complex. Considering Monobodies are classed as a mimetic, it is possible that the Monobody would insert into the crevice of the ICMT protein domain. Once there, the synthetic residues of the Monobody would interact with the residues of ICMT, forming hydrogen bonds with the residues between the spaces of M1-M8. The location of the Monobody in fact prevents the enzyme from interacting with its protein substrates as depicted by X-Ray crystallography (Diver, M. (2018). Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT. Nature, online 553(7689), p.1. Available at: https://www.nature.com/articles/nature25439 Accessed 31 Jan. 2018.). The enzyme keeps is distinct substrate specificity due to its positioning and shape but when exposed to the Monobody loses all specificity due to the hydrogen bonds disrupting the alpha helices and the Monobodies binding next to the active site. Nucleophilicity: A nucleophile is a substance that is nucleus-loving and attacks an electrophile which is electron-loving. In terms of ICMT inhibition, nucleophilicity would be an important aspect of inhibiting the enzyme as multiple residues are running throughout the structure. Using an electron rich atom that is seeking bond to reach the octet it is workable to inhibit the enzyme be destabilising the alpha-helical domains via nucleophilic attack on electrophilic amino acid residues (McMurry, J. (2018). Organic Chemistry. 9th ed. Cengage Learning, p.898.).Structural Mutations: ICMT in pat is structurally sound because of its eight alpha-helical domains. If certain helices were to be affected the enzyme will in turn lose all its enzymatic function. If helical domains M1 and M2 were to be genetically deleted (model completed in beetle genome) the enzyme is made completely inactive. In addition to this M4 and M5, when mutated via changed to their amino acid composition the both lose complete functionality. By destabilising the helicases proline rich connectors, the proteins lose all connectivity to one another in the overall structure. The reason for this importance as that all these features directly tie in with the active site and the cofactor pocket. The active site utilises most of the alpha-helices to stabilise its own structure and any mutations that affect the structure will in turn effect the active site. Therefore, there is two modes of inhibition here, one where the structure is destabilised but secondly the active site loses its ability to carry out reactions. This is especially true for the aromatic residues found throughout the structure.The cofactor AdoHcy and its modelled counterpart AdoMet, if mutated to Alanine it leaves ICMT with over ninety-five percent inactivity. The cofactor is essential to enzyme function so when it loses total functionality the enzyme itself will also lose its ability to catalyse reactions. Figure 5a: MonobodyThis is a figure which outlines the structure of a Monobody. Notice the difference between light and heavy chains including the brown structure which is called a FG loop. The FG loop is one of the key aspects of the Monobody as it plays a role in interacting with ICMT to inhibit it.Source: En.wikipedia.org. (2018). Monobody. online Available at: https://en.wikipedia.org/wiki/Monobody#/media/File:PDB_2rhe_EBI.jpg Accessed 30 Jan. 2018.Figure 5b: Nucleophilic AttackHere we see a generic nucleophilic attack. The Nu denotes the nucleophile which is electron rich as seen by added electrons on the outside of the Nu. It is attacking the Carbon atom which only has three bonds out of its required four showing its electron deficient. Once attacked the carbon shares the electrons with the nucleophile but due to its higher electronegativity will pull the electrons closer to itself. Source: Evenson, R. (2018). What is a nucleophilic attack?. online Quora. Available at: https://www.quora.com/What-is-a-nucleophilic-attack Accessed 30 Jan. 2018.Summary/Conclusion  To summarise the major findings of this article was the completed atomic structure of one of the most important enzymes that are a prevalent cause of RAS driven cancers and progeria. They utilised X-Ray crystallography as their main technique to figure out the structure/function relationship between mutation and inhibition. These results are novel in the way it is the clearest crystallised image of ICMT to date and this idea can be furthered to help in the long-term fight against aggressive cancers as seen in Discover where they look at mutations and genes in cancer therapies (Swartz, A. (2016). Fighting Cancer With Data. Discover, online (October 2016). Available at: http://discovermagazine.com/2016/oct/fighting-cancer-with-data Accessed 30 Jan. 2018.). A combination of these novel ideas might give the necessary knowledge to aid in clinical tests and trials. The next step for this article is to accurately test how inhibition affects ICMT.References1. Lodish, H. (2008). Molecular Cell Biology. 6th ed. W. H. Freeman, 2008, p.935.2. Lodish, H. (2008). Molecular Cell Biology. 6th ed. W. H. Freeman, 2008, pp.936-937.3. Diver, M. (2018). Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT. Nature, online 553(7689), p.1. Available at: https://www.nature.com/articles/nature25439 Accessed 26 Jan. 2018.4. Diver, M. and Long, S. (2014). Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites. Journal of Biological Chemistry, 289(38), pp.26007-26020.5. Yang, J. (2011). Mechanism of Isoprenylcysteine Carboxyl Methylation from the Crystal Structure of the Integral Membrane Methyltransferase ICMT. Molecular Cell, 44(6), pp.997-1004.6. McMurry, J. (2018). Organic Chemistry. 9th ed. Cengage Learning, p.898.7. Gao L, Liao J, Yang GY. CAAX-box protein, prenylation process and carcinogenesis. Am J Transl Res. 2009;1:312–PMC free article PubMed8. Wright, L. and Philips, M. (2006). Thematic review series: Lipid Posttranslational ModificationsCAAX modification and membrane targeting of Ras. Journal of Lipid Research, 47(5), pp.883-891.9. Lodish, H. (2008). Molecular Cell Biology. 6th ed. W. H. Freeman, 2008, p.542.10. Malumbres, M. and Barbacid, M. (2003). Timeline: RAS oncogenes: the first 30 years. Nature Reviews Cancer, 3(6), pp.459-465. 11. Solomon, H., Madar, S. and Rotter, V. (2011). Mutant p53 gain of function is interwoven into the hallmarks of cancer. The Journal of Pathology, 225(4), pp.475-478.12. Luo, J. and Elledge, S. (2008). Deconstructing oncogenesis. Nature, 453(7198), pp.995-996.13. Wang, T., et al. (2017). Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras. Cell, 168(5), pp.890-903.e15.14. Guttler, D. (2018). RAS Mutation in “benign”( NIFT-P)Non invasive follicular tumor with papillary cell features – Thyroid Center of Santa Monica. online Thyroidnosurgery.com. Available at: http://thyroidnosurgery.com/thyroid-cancer/ras-mutation-in-benign-nift-pnon-invasive-follicular-tumor-with-papillary-cell-features/ Accessed 30 Jan. 2018.15. Ibrahim, M., et al. (2013). Targeting Isoprenylcysteine Methylation Ameliorates Disease in a Mouse Model of Progeria. Science, 340(6138), pp.1330-1333.16. Johnson, T. (2013). Rapid Aging Rescue?. Science, 340(6138), pp.1299-1300.17. McMurry, J. (2018). Organic Chemistry. 9th ed. Cengage Learning, p.157.18. En.wikipedia.org. (2018). Monobody. online Available at: https://en.wikipedia.org/wiki/Monobody#/media/File:PDB_2rhe_EBI.jpg Accessed 30 Jan. 2018.19. Evenson, R. (2018). What is a nucleophilic attack?. online Quora. Available at: https://www.quora.com/What-is-a-nucleophilic-attack Accessed 30 Jan. 2018.20. Swartz, A. (2016). Fighting Cancer With Data. Discover, online (October 2016). Available at: http://discovermagazine.com/2016/oct/fighting-cancer-with-data Accessed 30 Jan. 2018.

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