The ubiquitin system, disease and drug discovery - Research Paper Example

?Ubiquitin function Ubiquitin is a protein comprised of 76 amino acids. Ub is a protein which is stable in heat that folds up into a globular pattern. Ub is found throughout an entire cell and can exist in a free state or combined with other protein. If it is combined with other protein then it is attached by a covalent bond between the glycine and lysine on the proteins. Ub has a role to play in many cell processes. For example – during the G1 phase of mitosis Ub is conjugated to the protein cyclin and hence play an important role in controlling the cell cycle. Ub conjugation is also present in DNA repair, the regulation of transcription, apoptosis and embryogenesis. According to (Hershko,1998,pg.425-79) “There are strong indications for roles of the ubiquitin system in development and apoptosis, although the target proteins involved in these cases have not been identified.”.Protein in ubiquting exists in chain format which is linear and consists of amino acids. The degradation of chain is possible and it is thermodynamically possible in an aqueous environment. When the degradation of protein happens this is known as protein – turnover. The balance which exists between the synthesis and its degradation determines the concentration level of protein in the cell. The studies conducted over protein turnover rates have revealed that some proteins are long lived while others are short lived. The cell majorly consists of long lived protein while short lived proteins which are regulatory protein are abnormal protein. Source – (Hersko,1998,pg 425-79) The Ub has a function to monitor the turnover of protein in the cell by regulating the degradation process. This regulatory function is largely important. By regulatory function of Ub the cells are able to eliminate protein that displays another function. Furthermore, such regulation observes that other process expressed by regulatory protein is shut down. There is another regulatory function displayed by the protein which is alternative in nature and it simply inactivates the protein. However, in case of this alternative regulation, these inactivated proteins can be mistakenly reactivated. Unfortunately, the Ub linked regulation is expensive energetically and if a regulation needs to be done once again then re-synthesize should be performed. The functioning of Ub is in a ATP – depended pattern. But what is the reason for this? The reason for this in order to target the protein machinery is required that can degrade the protein. The machinery is used just as a tag which marks the protein which needs to be degradation. The degradation is conducted by the 26Sproteasome. Speaking precisely, the proteins that are to be degraded are primarily tagged by conjugating them with ub and these tagged protein are them identified and shuttled to proteasome for the purpose of degradation. Dysfunction in much ubiquitin process has created pathological conditions where there was malignant transformation. Proliferation and cell growth are further controlled by ubiquitin mediated degradation of portooncogenes, tumor suppressors and components of signal transduction systems. The Ubiqutin – Proteasome Pathway If we mix ubiqutin, ATP and an abnormal protein we might think that the protein will conjugate with Ub. However, we would be wrong in this assumption. There is something else required to attach Ub to such a protein. The thing that is needs in many cases is the three kinds of enzymes. 1) Ub activating enzyme known as E1 enzyme. This enzyme is required in modifying Ub so that it can be in a reactive condition. 2) Ub conjugative enzyme known as E2 enzyme. This enzyme does the function of catalyzing the attachment of Ub to the substrate protein. 3) Ub ligases know as E3 enzymes. This enzyme function in align with E2 enzymes and this enzyme is important in identifying the substrate protein. Yeast contains many E1, E2 and E3 enzymes. For example. It has been found that yeast contains 13 various E2 enzymes. All these do the function of conjugation but also a specific function to perform. For example Ubc2 and E2 enzymes has a role to play in DNA repair while Ubc3 and E2 enzymes works to degrade cyclin as a process of cell cycle. Generally, the Ub is activated by E1 enzyme in an ATP dependant manner. Then the E2 and E3 enzyme helps in identifying the substrate protein which later conjugates Ub to it.Ub can be attached a synthesized chain or as a monomer. From this stage onwards, the ubiquinated protein is flown to proteasome for degradation. The Degradation Signals What do you think can determine the degradation of where the protein is tagged and heading for degradation? This answer to this question is just not possible yet but scientist has discovered some surprising clues about this process. Apparently, the protein comprises of some sort of signal which is identifiable by the Ub machinery. Some of the signals so far discovered are as follows; 1) The N- degron – Alexander Varshavsky is one of the renowned researchers in U b and in 1986 he conducted an experiment that exhibited a correlation between N- terminal residue of protein and half life of a protein. As per (Varshavsky,1996,pg.12142-49) “ The half- lives of proteins in a living cell range from a few seconds to many days”. The N- end rule is a phenomenon arose out of this observation where one gets the chance to predict the lifespan of a protein with the N- terminal amino acid. The recognition of the mechanism where the coupling of N- terminal amino acid and half life of protein occurs is still unknown. 2) The sequences of certain amino acids are an indication of degradation. One of such sequence is PEST sequence and here short stretch of around 8 amino acid is enriched with serine, glutamic acid,proline and threonine. The length of this protein is 281 amino acid and PEST sequence position is 91-106. The half life of protein is normally 5 minutes. Source - (Varshavsky,1996,pg.12142-49) 3) Many a time’s signals can also be hidden in hydrophobic core. This is the reason why abnormal or partially folded protein can get degraded. If such protein remains in native state the signals are not revealed and the protein tends to live for a longer time. But if the proteins are partially unfolded then the Ub machinery can detect it and the protein can be tagged. However, it is seen that chaperone activity hinders this activity. Ubiquination and Protein Degradation It should be noted that Ub itself does not degrade protein but only tag them for this purpose. However, it cannot be assumed that Ub is only a participant in tagging as it has a role to play in protein degradation process. The proteasome is what that really does the trick of degradation. As per (Low,2013,pg 1421-2121)“The proteasome is a multi-subunit protease complex, consisting of a 20S core particle and a 19S regulatory particle”. The role of the ubiquitine’s degradation may be to lower the disassociation rate between proteasomes and substrate proteins which interact. The protein in the absence of Ub quickly disassociate when they interact. The Ub has the function to slow down this disassociation process. A substrate protein which is conjugated with Ub interacts with proteasome for a longer period which enables the quick degradation of the protein. In real sense, the Ub has the ability to tie the substrate protein to the proteasome. Source –( Low,2013,pg.1421-2121) Pathway Description From yeast to mammal the ubiquitin proteasome pathway is required to target the protein degradation in most short lived eukaryotic cells. Cell cycle regulatory proteins are targeted whose destruction is timely fashion is needed for the vital cell division. Ubuiquiting modification is dependent on ATP. The ubiquitin pathway has been discovered in the 1930s and has played a big role in cell physiology in the last 25 years. According to (Mani,2005.pg.4776-4789) “Subsequent studies showed that degradation assumed equal weight as synthesis in determining the intracellular content of proteins”. The ubuiquitation machinery is present both in nucleus and cytosol. The degradation process is initiated by the E1 enzyme. The identity sequence of eukaryotic ubiquitins is same. The ubiquitization is a process which is steady and stable in nature and it can be reversed with the help of deubiquitinating enzymes. The degradation is initiated by a single enzyme E1 which activates the ubiquitin petide monomer. The proteasome which is a large subcellular organelle is a multisubunit protein complex and is a site for ATP dependant protein degradation. The function and structure of Proteasome is conserved in nature in eukaryotes and archaebacteria and proteasome is necessary for cell organism viability in eukaryotes. In eubacteria proteasome are not found. Ubuiqination is possible upon the binding of the target protein to the correct E3 ubiquitin ligase.The regulation of ubiquitin is also possible by the activation of certain E3 ligases. The ubiquitin degradation has been useful in treatment of many diseases from cancer to Alzheimer’s. Source – (Mani,2005, pg.4776-4789) Ubiquitin and Disease Proteasomes are complexes which are important for the body for the degradation of proteins that are abnormal in nature. For this very same reason, it has association with several diseases. A single complex is comprised of two enzymatic rings and two end caps structured in a barrel shape to enable their peptidase functionality. Abnormal proteins are the main reason for the onset of diseases in the body and with the proteasome function these are tagged and degraded before they can be identified by theproteasome cap. Once they are identified, the protesome system or UPS decomposes the abnormal protein. The utility and promotion of UPS plays a significant role in treatment of diseases like cancer, neurodegenerative disorders and other illnesses. As per (Ali,2008.pg.102.110) “The proteasome is a protein complex that bears the histological significance of numerous protein-related diseases. Among these are neurodegenerative disorders, prion diseases, and cancers”. Apparently, proteasome is beneficial in breaking down various abnormal proteins which is toxic to body. Ubiquitin proteasome pathway is paramount to this degradation process. For many years, the medical experts thought the degradation process occurred only by lysosomes. However, a recent discovery has established that the real truth behind degradation where the ubiquitin and proteasome work together in a process to tag and degrade the abnormal proteins. In disease process the ubiquitin process malfunction and proteins which are defective in nature are not decomposed. Source -(Ali,2008.pg.102.110) The ubiquitin proteasome pathway and its contribution to cancer cell dissemination has been an important issue for many years. As a single agent an inhibitor called bortezomib played an important role in inhibiting tumor growth but with the combination of chemotherapeutic agents there was anti tumor activity present.Ubiquitylation is a mechanism which is newly used to in variety of non –proteolytic cellular function. In breast cancer, several drugs that stimulate proteasome pathway for the degradation of the diseased protein have been evaluated. However its effect on the cancer treatment is not yet known. In his article ( Orlowski,2003,pg .1-7) writes that “Proteasome inhibitors may also be effective in breast cancer treatment by helping to overcome some of the major pathways by which cancer cells resist the action of chemotherapy”. Source – (Orlowski,2003,pg .1-7) Ubuiquitin is also seen to be useful in treating Alzheimer’s disease which is most prevalent among aged people in Western countries. It is a neurodegenerative disease which has an unknown etiology. The patients depict the formation of amyloid fibres which can be contributed due to the ubiquitin mediated degenerative defects. The studies have reveled that UPS needed for protein repair degradation and turnover is disturbed in the patients. It is understood that protein damage can be caused due to protein oxidation which result in dysfunction of proteasome. In neurodegenerative disease accumulation of protein is a recurring event and this happens due to the dysfunction of proteasome. In Alzheimer’s disease there is evidence of biochemical and genetic involvement which lead to ubiquitin proteasome dysfunction .Alzheimer’s is a neurodegenerative disease where the patient experience loss of memory, severe dementia and cognitive functioning problems. In Parkinson disease also there has been evident proteasome dysfunction and there was seen mutation in the proteasome function of the parkin gene. It is found that mutation of synuclein and parking gene is the reason for the onset of Parkinson syndrome. Ubiquitin is present in all cells of the body and it is most seen protein in the brain. If the ubiquiting proteasome system is defected then the protein molecules tend to get accumulated in the cell which interfere with the normal functioning of the cell functioning and can also result in damage or destruction of the cell. Genes are which regulates the ubiquitin synthesis an d scientist have found many mutation in the gene that regulates the ubiquitin in people suffering from neurodegenerative diseases such as Parkinson’s and Alzheimer’s. The protein accumulated in these disease conditions is called Tau in Alzheimer’s and Alpha – synuclein in Parkinson’s disease. The ubiquitin that regulate use and production of ubiquitin is exist in close region to the gene that control various protein function connected to Parkinson’s. Scientist suspects that mutation of these genes has connection to the malfunctioning of all of these proteins. However, other scientist believes that ubiquiting gene mutation only disable the tagging and degradation of protein which allows the accumulation of Tau and Alpha- synculei protein in the cell. According to (Mosley, 2010,pg.307) “Researchers believe when the ubiquitin proteasome system malfunctions, damaged and ineffective protein molecules remain within the cell”. These lumps of molecules damage the functionality and cell and can lead to diseases. Source - (Mosley, 2010,pg.307) Ubuiquitin and Drug discovery In the ubiquitin system, the 206 proteasome is the only validated therapeutic target with a sole commercially available drug called Velcade. Currently the drug discovery activities related to Ubiquitin centers around three areas: a) enhancing signs of inhibition of proteasome in therapy (b) developing proteasome inhibitors with targeting ability with increased bio – availability (c) Validating the potential of other targets of ubiquitin system. The diversion in the functions of proteasome has been useful in treatment of various diseases. In 2003, the drug Velcade which is a molecule proteasome inhibitor has been approved by FDA for the treatment of relapsed multiple myeloma.Nowadays, many experiments are being conducted to develop inhibitors which can target distinct activities of the proteasome. Proteolix is a company which has discovered PR – 171 a synthetic analog of epoxomycin which can irreversibly inhibit the site of proteasome. Experiments have shown that PR – 171 has a more prominent anti tumor effect than Velcade.Another drug which is in initial phase of experimentation is salinosporamide invented by Nereus pharmaceuticals. This medicine has more inhibition properties than Velcade. It is also has the capability to respond well to the multi – drug treatment related to colon cancer. These two drugs mentioned are known for it’s therapeutically value in protesome inhibition. Other developing drugs areNovartis AG,Bristol- Myers Squibb,Cytomics Systems and Cephalon. According to ( Petroski,2008,pg. 112-20) “The development of inhibitors that disrupt this interaction will play a major role in the regulation of cell cycle progression and potentially the treatment of cancer”. Source -( Petroski,2008,pg. 112-20) Recently, there has been evidence for the pharmaceutical efficacy of proteasome in the treatment of human cancer. The cell signaling is an important part of protein regulation and it is done with the help of protein turnover and it is also called protein phophorylation. In diseases, the role of ubiquitin is in the infancy stage and inhibition of the proteasome is the clinical way to treat diseases.Bortezomib a peptide boronic acid inhibitor of the proteasome is the drug that primarily approved for human use which can target UPS. The potentiality of the drug to kill the tumor cell with a satisfactory side effect was the surprising feature of this drug in the initial phase. However, now it has been found that the tumor cells generate higher oncoproteins and aberration proteins which make inhibition of proteasome more sensitive. The success of inhibition of proteasome largely depends on development of more compounds targeting UPS which can treat certain types of cancer. When developing drug for the UPS several issue need to be considered by the researcher. Firstly, the type of the disease that is been targeted and what is the alteration in pathway indented? Secondly, how particular types of protein within the UPS can be targeted? The potential site for drug development is the interaction between E1 and E2 enzymes. It is important to understand that such protein interaction can make novel drug innovation difficult due to the difficulty in identifying the small – molecule binding pockets. According to (Patterson, 2005,pg. 3) “ Proteasome holds a unique position among intercellular proteases”. Source - (Patterson, 2005,pg. 3) Recently oxford inventors have created an ubiquitin like active site probes which can be changed to with the help of click chemistry to add functional features as fluorescent tags. These probs give way to many advantages in profiling DUBs in intact cells and cell extracts. This strategy requires minimum protein modification leading to an easy and affordable synthesis route. This click chemistry also allows adding additional features which diversify and expand ubiquitin based probes. Activity based ubiquitin probes are engineered specifically to mimic natural substrate enabling researchers to recognize, characterize and isolate these enzymes. The broad spectrum of UPS enzymes and substrates give way to even the treatment of diseases like HIV. One are which promise in drug discovery is the development of agents that inhibit Nedd8 E1. This Nedd8 based discovery has the potentiality to alter the function and fate ofcullin RING substrate. An agentMLN4924, is now under clinical trial for hematological malignancies and solid tumors. Other than proteasomes and enzymes, chaperones and ubiqutin receptors are other promising agents. One of the notable among these is hexameric for which inhibitions has been recently developed. The target – rich ubiquitin system has the therapeutic advantage of enhancing the personalized medicine and treat diseases which were previously untraced. The research field is trying to understand more about proteasome as a molecular machinery of ubiquination. As per (Acton,2013,pg .71)‘According to News reporting out of Paris, France by NewsRx editors, research stated, The ubiquitination- proteasome degradation system is an essential process that regulates the protein homeostatis”. Source - (Acton,2013,pg .71)‘ One of the biggest challenges faced by researchers in the field of drug discovery related to ubiquitin is that ubiquitn ligases and enzymes of interests present difficulties in targeting specific ligase inhibitors. The researchers are understanding the ubiquiting ligase functions and their physiological relationship with protein substrates and other ubiquiting enzymes which can lead way to target its activities.In Yale University several studies are conducted regarding ubiquitin proteasome system in the department of bio chemistry.According to the experts lot need to be learnt regarding de – ubiquitinating enzymes and their substrates and about proteasome also. The major area of discovery is active site inhibitors in the therapeutic invention field. Since there is lot of lab work occurring related to proteasome there is a possibility of new class of pharmaceutical targeting proteasome can be discovered. Various companies like Boston Bio chem.,Calbiochem, and Enzo life science working with protesome system has offered various tools incuding ubiquitin, relevant enzymes, antibodies, inhibitors, modified ubiqutines and substrates. For those experts studying the regulatory factors of the proteasome system can use these tools to enhance their experimental value. For example. PCR array for profiling the expression of 84 genes in the protesome system can easily enable researchers to analyze the expression of gene involved in ubiquitin degradation. Despite all the odds, the scientific field have discovered therapeutic agent Velcade, a proteasome inhibitor.De- ubiquinating enzymes are also promising but more need to be understood regarding their functioning. According to (Kyprianou,2012,pg.114) “ The clinical presence of the proteaome inhibitor botezomib ( Velcade) is rigorously established”. Source - (Kyprianou,2012,pg.114) Conclusion Ubiqutin being a main protein in all cells of living beings, the future prospects of this system in human diseases and drug discovery is major. However, we face a large gap in our knowledge about ubiquitin system where different types of ubiquitin ligases are unknown as opposed to the enzyme’s role in selectivity and regulation of ubiquitin mediated protein degradation. As many degraded are recognized by different E3 enzymes, the number of E3 enzymes is likely to be greater than E2 enzymes. At present, the cloning has been done of yeast E3 a and E6 – Ap like E3 enzymes. Based on the developments occurred in past , the assumption is that ubiquitin field will continue to expand rapidly, basically by three types of approaches. As per (Peters,1998,pg.22) “Ubiquitin genes are unique , in that all of them specify fusion between ubiquitin and other polypeptides, including other ubiquitins “. One is the by realizing that ubiquitin mediated degradation of key regulatory proteins has great role in cell cycle. In this specific case, the challenge will be to understand degradation function of the regulatory protein.Secondly, investigators from other field may attempt to clone a gene possessing an interesting function then by homology sequence finding the family belonging of the gene with regard to ubiquitin enzyme. In third approach specific inhibitors are utilized which implicates the ubiquitin system functioning in antigen presentation and apoptosis. Finally, it should be given attention in future research on the elucidation of some ubiqutin processes that do not give way to degradation by 26 S proteasome. Source - (Peters,1998,pg.22) The main issue that can be imbibed from the brief evaluation of the past and present development in the field of ubiquitin is the importance of molecular genetics and biochemistry in future progression in biological research. In the absence of biochemistry, the mode of action of various genes of the components of ubiquitin system in relation to cloning can be still a mystery. On the other hand, molecular genetics is necessary to reveal the great variety of basic cellular function of ubiquitin system. Until recently, the scientific world thought of ubiquting system as a “garbage disposal” for the discarding of abnormal proteins. This assumption is definitely not true for those of us understanding the importance of selective degradation of protein in cellular functioning. It is a fact that without molecular genetics, it is not possible for us to understand the ubiquitin protein degradation process and its essential functions. So it is assumed that biochemistry combined with molecular genetics approach will lead to many advances in future in the ubiquitin field. However, the therapeutical benefit of ubiqutin pathway in cancer treatment has been a bigger breakthrough in research field. Due to the deregulation in cancer developments, concentrating on ubiqutin system in cancer therapeutics emerge as a promising approach. However, the challenge is in finding drugs which act specifically on ubiquitin substrate which work without affecting other pathways. As per (Haendler,2009,pg.24) “ In general structural studies of 26S proteasome are hampered by their low intrinsic stability,, leading to disassociation in to various subcomplexes”. Source -(Haendler,2009,pg.24) Since ATP – dependant involvement is found in ubiquitin activation and proteasomal degradation and they represent classic drug targets, it is an attractive perspective for therapeutic al intervention. However, the major concern related to the wide action on various substrates and pathways can give way to side effects. Fortunately, the biggest breakthrough in targeting the ubiquitin system in cancer therapy has been the discovery of Bortezomib a proteasome inhibitor.Botezombic shows selective cytoxity against cancer cells in comparison to normal cells. Therefore development strategies show proteosome inhibitors useful in certain kinds of tumors. Other than targeting ubiquting system itself, some clinically effective antibodies and molecule inhibitors have shown to enhance degradation process. The use of Herceptin, an anti body used for the treatment of breast cancer tumors has been useful in degradation. Another challenging and promising approach in ubiquitin related cancer therapy is to alter the protein interaction induced by ubiqutin.The degradation process of ubiquitin system has been much studied. As there are many cellular protein turnovers taking place, the exact mechanism that regulates the function is not precisely known. The ubiqutin has role in many cellular functions and the proteasome activity in relation to chemotherapeutic agents has led to the conferring of Nobel Prize for discovery of ubiquitin. As per (Deshaies,2005,pg.110)“ The award of the 2004 Nobel Prize for Chemistry to the founders of the ubiquitin Proteasome system of intracellular proteolysis is a timely importance of the UPS of the cell physiology and disease pathogenesis” Source - (Deshaies,2005,pg.110)“ Since the scientific field has revealed the therapuetical value of Ubiqutin, big pharmaceutical companies have shown interest in ubiquitin researches in future. The industry has been convinced that Ub has a great potential in drug discovery and many research projects related to it is in pipeline. Though ubiquitin research is a confined community, the diversity and growth in this field shows that it has a huge hope in future. However, further technological trends and innovation can bring about more developmental trends. It is astounding that the research regarding ubiquitin has gone beyond cancer and neurodegenerative diseases. With its potentiality for drug discovery it has awakened much interest for public health benefits. Research on ubiqutin has revealed that it has potential to fight many immunity disorders. The Velcade and Bortezomib are major drugs which are successful in treatment of cancer. The future research is focused on the recognition of new target proteins by the system. Up-to-date, the study and research about ubiquitin proteasome system represents one of the most challenging fields in applicable science. The ubiquitn ligases and and proteasome function is a major focus of drugs and pharmaceutical companies. According to (Grottreup,2011.pg 123) “ Since genetic evidence is largely missing, the actual importance of the individual ubiquitin ligases for P53 ubiquination and degradation remain to be determined”. However, after all the study in the field of ubiquitng there has been delay in the drug screening and development process. Source - (Grottreup,2011.pg 123) Bibliography Acton, A. (2013). Aromatic Amino Acids—Advances in Research and Treatment: 2013 Edition (p. 71). Georgia: Scholarly Edition. Ali. M (2008) .The Role of Ubiquitin-Proteasomal Pathways in Specific Disease Processes. The Georgetown Undergraduate Journal of Health Science, 5(2), 102-110. Deshaies, R. J. (2005). Ubiquitin and Protein Degradation (p. 110). London: Elsevier Academic Press. Groettrup, M. (2011). Conjugation and Deconjugation of Ubiquitin Family Modifiers (p. 123). New York: Springer. Hershko, A.(1998). THE UBIQUITIN SYSTEM. Annual Review Biochem, 67, 79-425. Haendler, B. (2009). The Ubiquitin System in Health and Disease (p. 24). Heidelberg: Springer- Verlag. Kyprianou, N. (2012). Molecular Exploitation of Apoptosis Pathways in Prostate Cancer (p. 114). London: World Scientific Publishing Co. Low, P. (2013). Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila. BMC Cell Biology, 14(29), 1471-2121. Mani, A. (2005). The Ubiquitin-Proteasome Pathway and Its Role in Cancer. Journal of Clinical Oncology, 23(21), 4776-4789. Mosley, A. D. (2010). The Encyclopedia of Parkinson's Disease (2ndnd ed., p. 307). New York: Infobase Publishing Inc. Orlowski, R. Z. (2003). The role of the ubiquitination-proteasome pathway in breast cancer: Applying drugs that affect the ubiquitin-proteasome pathway to the therapy of breast cancer. Breast Cancer Researc, 5(1), 1-7. Patterson, C. (2005). Ubiquitin-Proteasome Protocols (p. 3). New Jersey: Humana Press Inc. Petroski, M. D. (2008). The ubiquitin system, disease, and drug discovery. Bio Med Central, 9(1), 112-120. Peters, J. M. (1998). Ubiquitin and the Biology of the Cell (p. 22). New York: Plenum Press. Varshavsky, A. (1996). The N - End rule: Function, mysteries , uses. PNAS, 93, 12142-12149. Read More