proteasome assignment

8/13/2019 Proteasome Assignment

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2013/2014

Histology Department

[PROTEASOME] Done by: M arin a, Maryam, M ai, Ayesha, M ohamed


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Introduction:Proteasomes are non-membranous organelles which are ATP dependant

protease complexes that represent 1% of cellular protein. They are inside alleukaryotes, archaea, and in some bacteria.

They function to degrade, denatured or otherwise nonfunctional polypeptides; Onething that is known is that in general, the cell is a very inhospitable environmentfor proteins, giving way for a multitude of errors. For example, the temperature ofthe cell is of 37C or higher, which can easily denature a protein. Denatured

proteins may have a hydrophobic area, which marks them to be broken down.Proteins that are less stable, for example, proteins with an exposed amino acidsequence that is unusually prone to hydrolysis, are more likely to be tagged fordegradation. Some proteins, such as cyclins, commonly contain a destructionsequence in order for them to be sent to the UPS (a system for maintaining the

protein concentration in the cells). There are many other molecules in the cell thata protein may attempt to react with, causing oxidation, deamination, glycation, ornitrosylation. There are other enzymes within cells, such as proteases or kinases,which may modify the protein. The salt concentration and fatty acid concentrationmay also denature or dissociate the proteins. With all of these possibilities forerror, it is clear how a protein may be altered very quickly following its synthesis.Up to 80% of the proteins in a cell may be abnormal and they must be destroyed

before they can cause harm to the cell, so Proteasomes are essential to thedegradation of abnormal proteins, and are thus, naturally tied with several protein-related diseases, Among these are neurodegenerative disorders, prion diseases, andcancers, we will talk about it in more details later.

Site : In eukaryotes, they are located in the nucleus and thecytoplasm, not associated with membrane, each approximately the

size of the small ribosomal subunit. Structure : The proteasome is a cylindrical complex containing a"core" of four stacked rings around a central pore. The inner tworings are made of seven subunits that contain three to seven

protease active sites. These sites are located on the interior surfaceof the rings, so that the target protein must enter the central pore
http://en.wikipedia.org/wiki/Protein_complexhttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Archaeahttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Cytoplasmhttp://en.wikipedia.org/wiki/Cytoplasmhttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Archaeahttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Protein_complex


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Figure (1): showing the structureof the roteasome.

before it is degraded. The outer tworings each contain seven subunitswhose function is to maintain a "gate"through which proteins enter the

barrel. These subunits are controlled by binding to "cap" structures orregulatory particles which are at theend of each cylinder that recognize

polyubiquitin tags attached to proteinsubstrates and initiate the degradation

process. The overall system ofubiquitination and proteasomal

degradation is known as the ubiquitin- proteasome system. (1)

Proteasomes are part of a major mechanism by which cells regulate theconcentration of particular proteins and degrade misfolded proteins taggedwith ubiquitin. The degradation process yields peptides of about seven toeight amino acids long, which can then be further degraded into aminoacids and used in synthesizing new proteins. They are essential for manycellular processes, including the cell cycle, the regulation of geneexpression, and responses to oxidative stress.

H istological pictur es:

L M :

Proteasomes are seen by using the Immunofluorescence techniquewhich is based on the use of specific antibodies which have beenchemically conjugated to fluorescent dyes. (2)


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EM:

TEM images of proteasome have been produced from many differentcell types. 3D image reconstructions reveal the molecular detail, with anaverage length of ~ 45 nm and diameter of ~13 nm . A long channel with adiameter of ~ 6nm (resembling a pore when seen from above) is located inthe centre of the cylindrical molecule. Higher resolution X-ray structuraldetail is also available. (3)

Figure (2): showing the LM picture of proteasomes


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Function:

For years, protein degradation was thought to occur only bylysosomes. But a recent discovery has revealed the truth behind proteindegradation within a cell: that proteasomes and ubiquitin actually worktogether in a system to tag and destroy abnormal proteins. Whereaslysosomes digest bulk material introduced into the cell, or wholeorganelles and vesicles, proteasomes deal primarily with proteins as

individual molecules. The first step in the process requires for there to bean abnormal protein, and proteins may be labeled abnormal for variousreasons. There are several reasons a protein may be destined fordegradation within the context of everyday metabolic activities inside thecell. The cell may produce proteins that have misfolded or contain errorsin the peptide sequence, and it is beneficial and necessary to the health ofthe cell that these proteins be degraded. Some of them do not fold

properly, like in insulin. Some proteins may be incomplete strands due to

an error in RNA splicing. Some of this could be caused by transcriptionand translation errors, but the exact reasons are still unknown.

1. Main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes thatcarry out such reactions are called proteases. Protein degradation is as essentialto the cell as protein synthesis, For example:

Figure (3): showing the EM picture of proteasomes


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Figure (4): showing the ubiquitin- proteasome system.

To supply amino acids for fresh protein synthesis. To remove excess enzymes, proteins encoded by viruses and other

intracellular pathogens. To remove transcription factors those are no longer needed.

2. They play an important role in the immune system by generating antigenic peptides that are presented by the major histocompatibility complex (MHC)class I molecules.

3. Ubiquitin-mediated proteolysis is one of the key mechanisms underlying cellcycle control. The removal of barriers posed by accumulation of negativeregulators, as well as the clearance

of proteins when they are no longerneeded or deleterious, are carriedout via the ubiquitinproteasomesystem.

-Ubiquitinis an abundant cytosolic76-amino acid protein found in allcells and is highly conserved

during evolution it has virtuallythe same structure from bacteria to humans. Denatured proteins or proteins withoxidized amino acids are targeted fordestruction after recognition byenzyme complexes which conjugate amolecule of ubiquitin to a lysine residue in the protein, followed by formationof amultiubiquitin chain. An ubiquinated protein is recognized by the regulatory

particle of proteasomes, unfolded by the ATPase using energy from ATP, andthen translocated into the core particle, where it is broken into short peptides.These peptides are transferred to the cytosol and the ubiquitin molecules arereleased by the regulatory particles for reuse. The peptides may be broken downfurther to amino acids or they may have other specialized destinations, such asthe antigen-presenting complexes of cells activating an immune response.


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4. DNA damage triggers polyubiquitylation and degradation ofthe largest subunit of RNA polymerase

II (RNAPII), a mechanism of lastresort employed during transcriptionstress (4) .00000000000000000000000000000000000000000000000000000000000000000000000000000000000

5. Proteasome plays a pivotal role in theselective recognition and degradation

of oxidized proteins produced from aerobic metabolism and environmentalstressors. These proteins may become cytotoxic thus; Cell homeostasis andviability are dependent on the removal of oxidatively damaged proteins.

Given that many proteins targeted by proteasome are involved in theregulation of important processes of carcinogenesis and cancer cellsurvival, such as cell cycle progression, cell proliferation, differentiationand apoptosis, inhibition of proteasome would lead to cell death orapoptosis.

Clini cal and M edical appli cation : In disease processes, the ubiquitin tagging mechanism malfunctions, and

defective proteins are not degraded by the proteasome. If ubiquitin receptor proteins do not first unfold and refold defective proteins, the proteins are taggedwith ubiquitin and sent to the proteasome for degradation. The ubiquitin-proteinligase is often defective during diseases processes, and the proteasome has noindication that the proteins need to be degraded. This can lead to an accumulationof defective protein aggregates, which raise the toxicity of the cell and may lead torapid cell death. In some cases, one of these insoluble aggregates will block the

proteasome from performing its function, which also leads to a build-up ofdefective proteins. In a third situation, aging proteasomes may simply slow down


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This accumulation of unwanted proteins due to the defective proteins isrelated to the Alzheimers disease .

The proteasome as a drug target, Proteasome inhibitors were initiallysynthesized as in vitro probes to investigate the function of the proteasomescatalytic activity. As the essential role of the proteasome in cell function wasclear up, the possibility that proteasome inhibitors may have potential astherapeutic agents was considered. Further in vitro investigationsdemonstrated that proteasome inhibitors displayed a broad spectrum anti-

proliferative and pro-apoptotic activity against haematological and solidtumors .

Cri tical targets for proteasome in hibitors in mali gnant cell s ; Pre-clinicalstudies have demonstrated that malignant cells are more susceptible to thecytotoxic effects of proteasome inhibition than normal cells. Themechanisms behind the higher sensitivity of malignant cells are unclear;however, it is likely that they use the proteasome to regulate proliferationand anti-apoptotic pathways. Most tumor cells are highly proliferative andhave an increased requirement for protein synthesis which would make themmore vulnerable/exposed to proteasome inhibition.

Proteasomes Role in Cancer ; Proteasomes also play a role in the disease processes of cancer. The role of proteasomes is complicated by apparent pro- and anti- apoptotic effects of in cell growth and development. As

previously described, proteasomes are highly effective in their destruction of proteins. Apoptosis, defined as a genetically determined process of cell self-destruction that is marked by the fragmentation of nuclear DNA, is activatedeither by the presence of a stimulus or by the removal of a stimulus orsuppressing agent. Apoptosis requires each cell to carry the proteinsnecessary for its death. An ubiquitin-proteasome system helps prevent

premature cell death by destroying proapoptotic proteins. Control of proapoptotic proteins ensures the prevention of abnormal activation of cellapoptosis. Unfortunately , these processes can be used by viruses to theirown advantage. One such case is the potency and prevalence of HumanPapillomaviruses. HPV is linked to genital warts, as well as anal andcervical cancers. These cancerous growths, under normal conditions, would

be blocked by p53, a tumor suppressor. However, the gene product E6 ofHPV targets the p53 protein for degradation by the ubiquitin-proteasomesystem. This is accomplished by making a protein that binds simultaneouslyto p53 and an E3 protein (responsible for marking proteins as targets for


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ubiquitination). The ubiquitination of p53 leads to its degradation by the proteasome. An additional factor, E6-AP (associated protein), was also proven necessary for the E6-dependent ubiquitination of p53. E6-AP, anubiquitin-ligase, suggests a cascade pathway including E1-E2-E3 necessaryfor ubiquitination.

In addition, proteasomes, or rather the inhibition of proteasome complexes, play an instrumental role in the induction of apoptosis. These effects arefound in rapidly growing cells. In response to apoptotic stimuli, threesubunits of 19S are cleaved by caspases and are, therefore, unable to destroyubiquitinated substrates. Ubiquitinated proteins accumulate and lead to anincrease in the levels of proapoptotic proteins, resulting in not only a criticalthreshold for caspase activation, but also intensification the apoptotic signal.For this reason, the use of proteasome inhibitors in the promotion of cellapoptosis plays a pivotal role in the treatment of cancers.

In a clinical setting, cancer treatment involves inhibition of the UPS bymedications that interact with the proteasome complex. However, thefunctions of these medications counteract treatments for neurodegenerativediseases, as proteasome inhibition can significantly affect proteinopathies.To combat this obstacle, proteasome-inhibiting medications have beendesigned to avoid the entering into nervous system.

Videos:For understaning better,

http://www.youtube.com/watch?feature=player_embedded&v=4DMqnfrzpKg http://www.youtube.com/watch?v=hvNJ3yWZQbE

Summary:

Proteasomes are complexes in the body essential to the degradation of abnormal proteins, and are thus, naturally tied with several disease processes. A singlecomplex consists of two end-caps and two enzymatic rings, stacked in a barrel-shaped manner to facilitate their form with peptidase functionality. Abnormal

proteins in the body, often precursors to disease, are first tagged to be degraded bythe protein ubiquitin before they can be recognized by the proteasome cap. Once
http://www.youtube.com/watch?feature=player_embedded&v=4DMqnfrzpKghttp://www.youtube.com/watch?v=hvNJ3yWZQbEhttp://www.youtube.com/watch?v=hvNJ3yWZQbEhttp://www.youtube.com/watch?feature=player_embedded&v=4DMqnfrzpKg


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recognized, the ubiquitin-proteasome system, or UPS, ultimately decomposes theabnormal proteins into chains of amino acids as they move along the internal core.The promotion and utility of UPS plays an essential role in disease processes suchas neurodegenerative disorders, cancers, and prion-related illnesses.

Neurodegenerative diseases are characterized by clumps of accumulated abnormal proteins that are unable to be broken down by the UPS. Similarly, prion-relateddiseases employ a mechanism that triggers abnormal aggregation of proteins,impairing the proteolytic activity of the UPS. Within the context of cancers,

proteasome promotion and inhibition both play instrumental roles in the clinical prognosis. Potent viruses, such as HPV, have adapted ways to fool proteasomesinto destr oying the bodys own benefactors, tumor suppressor proteins, thereby

prevailing over the cells proteolytic defenses. The extent of the faculties of theUPS and its roles in treatment and prevention of diseases, specifically entailingthose mentioned, remain the foci of contemporary biomedical research.

References:1. Junqueiras Basic Histology book, Text & Atlas2. Structural Studies of the Proteasome & Implications for Proteasome

Activation by Eugene I. Masters3. Electron Microscope Atlas of Cells4. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Proteasome.html5. https://blogs.commons.georgetown.edu/journal-of-health-sciences/issues-

2/previous-volumes/vol-5-no-2-december-2008/the-role-of-ubiquitin- proteasomal-pathways-in-specific-disease-processes/

6. http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Proteasome.html7. Wilson MD, Harreman M, Taschner M, Reid J, Walker J, Erdjument-

Bromage H, Tempst P, Svejstrup JQ. (2013). Proteasome-mediated processing of Def1, a critical0step in the cellular response to transcriptionstress. Cell. 154(5):983-95. PMID:23993092

8. Peters, Jan-Michael; Franke, Werner W.; Kleinschmidt, Jiirgen A. (March

1994). "Distinct 19 S and 20 S subcomplexes of the 26 S proteasome andtheir distribution in the nucleus and the cytoplasm". The Journal ofBiological Chemistry 269

9. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088792/10. Wikipedia.com
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088792/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088792/


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