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Within the past few years, the knowledge and amount of research about the ubiquitin proteasome system (UPS) has expanded exponentially. With the numerous advances in technology and medical research, researchers can now start to focus on the physiological aspects of the UPS and how it relates to various physiological conditions within the cell. The main proteolytic component that comprises the UPS is the proteasome. Utilizing the proteasome, the UPS carries out numerous tasks that include the maintenance and regulation of basic cellular processes such as differentiation, proliferation, cell cycle, and apoptosis (cell death). The UPS is a major protein degradation machine within the cytosolic and nuclear regions of the cells that removes the majority of unneeded proteins that have fulfilled their obligations and also the damaged proteins via numerous proteolytic enzymes (Wang et al 1). The UPS is important for many important health-related concerns throughout entire body.
Recent studies have revealed that UPS dysfunction is associated with many forms of human diseases, such as neurodegeneration, heart failure, and various forms of cancer. The proteasome can be involved in immunological functions, and it has adapted to efficiently defend against various stresses that the cells encounter throughout the course of their lives. However, the UPS can also be paradoxical in its nature of functioning under stress; while its role may be beneficial in certain diseases, in others its role can be detrimental. Using cardiomyocytes, or heart muscle cells, the regulations of proteasome function can be found by utilizing protein kinase C (PKC) with a UPS function reporter. The testing of this theory will show a quantifiable effect on the inhibition or stimulation of the PKC signal transduction pathway with UPS.
The theory that PKC signaling is involved in regulating the UPS function remains untested in any forms of studies. Through personal experimentation under the supervision of Drs. Huabo Su and Xuejun Wang at the Division of Basic Biomedical Sciences of the University of South Dakota, other researchers and I will perform primary research within this new line of molecular science. Dr. X. Wang's lab will conduct studies that examine the effects of PKC alterations on the UPS function, using the reporter cell line that stably expresses modified Green Fluorescent Protein and Red Fluorescent Protein (GFPu/RFP). The ratio of GFPu/RFP can be used as a reliable readout for proteasome function. Using the most recent research advances with UPS and looking into side effects associated with alterations of the PKC signaling pathway, future research will determine the various effects that PKC has on proteasomal proteolytic function in cardiac cells. If valuable insight is gained from studying the effects that PKC may have on the UPS, then completely new studies could be developed in order to combat many diseases. Medical science can gain valuable insight into the understanding of the proteasome by increasing the research into the unknown role of protein kinase C and how it may affect the homeostatic nature of the cell.
Before discussing the possible interactions with UPS and PKC, one must first understand how the cell creates proteins and why the complex process is beneficial to the cell. The ability of proteins to function correctly in the cell is an important field of research in all lineages of medicine. In general, proteins are used for both structure and function in a cell, which in turn maintain the regularity of the organism provided that the path from creation to utilization is carried out with little or no problems. Knowing how proteins are made from the beginning to end allows individuals to understand the important detail and the enormous task that the cell undergoes to carry out its functions.
Within a basic cell there are many parts that ensure the survival of that cell and the organism that it supports. Proteins are defined as "One of the most fundamental building substances of living organisms. A long-chain polymer of amino acids with twenty different common side chains, occurs with its polymer chain extended in fibrous proteins, or coiled into a compact macromolecule in enzymes and other globular proteins" (Sadava). The research done on proteins is important because it enhances understanding of how these important building blocks function and what happens when proteins do not work properly. Cells are made up of many parts that move about inside of a thick fluid environment that has a viscosity similar to pudding known as the intercellular matrix, or cytosol. In this environment, these cellular components interact to create proteins and carry out the processes that maintain the cell's structure and function. The cellular part/organelle that plays a major role in creation of new proteins is the ribosome, and it symbiotically works together with other organelles to ensure the survival of the organism. The ribosomes construct the amino acids into a polypeptide chain by the processes known as transcription and translation. A better understanding of the manufacturing of proteins is essential to grasp why the UPS is important for cellular survival.
The central dogma of molecular biology is that proteins are created from DNA. To get from DNA to RNA, and then proteins, the cell must go through multiple processes. Transcription is known as "the synthesis of RNA using one strand of DNA as the template" (Sadava). The creation of protein with RNA happens in three phases called initiation, elongation, and termination. DNA is short for deoxyribonucleic acid and is made of the nucleic acid bases adenine, cytosine, guanine, and thymine. The four nucleic acids are arranged in a pattern that encodes all of the genetic information necessary for life; in order to encode proteins, RNA (ribonucleic acid) must be used to transcribe a duplicate complimentary template. Transcription is also referred to as the expression of the gene. The enzyme RNA polymerase works at a specific region known as the promoter to unwind the DNA and make the new complimentary template strand. Next the polymerase continues along the strand copying one side of the DNA, and it copies the bases into the RNA components adenine, cytosine, guanine, and uracil, with uracil replacing the original thymine bases from the DNA strand. This process, called elongation continues along the DNA strand making a complimentary RNA strand, rewinds the separated DNA, and finishes at the termination site (Sadava et al 258-265).
The "code" in the RNA strand makes proteins in a process known as translation. The "code" in the protein is the result of three-letter pairs of RNA that form triplet bases called codons. This is done using messenger RNA (mRNA), and these codons create amino acids inside of the ribosome. Acting as a factory for the creation of proteins, the ribosome is the site of translation and it is similar to transcription in that it is also comprised of three steps: initiation, elongation, and termination. Amino acids are linked together by the mRNA and transfer RNA (tRNA) by interacting with the ribosomes to create the polypeptide sequence (Sadava et al 268). Throughout these steps the strand of the mRNA creates a polypeptide chain that grows longer and is secreted outside of the ribosome into the cytoplasm as it elongates. From the cytoplasm, the secreted polypeptide is shaped into a protein that can go to multiple sites necessary for "posttranscriptional modification," which further modifies the protein for its unique function (274).
Cells are constantly creating a wide variety of proteins. The sequence of codes taken from the DNA or RNA needs to be read correctly and also folded into the correct shape to ensure that no harmful mutations occur within the cell. In relation to speed, increased stress on the cell can lead to damaged proteins. The cells of an organism must go through growth cycles and completely duplicate their DNA while simultaneously preparing all of the other proteins necessary for the duplication. The creation of proteins is governed by how fast the DNA encoding the protein can be converted into mRNA, and then the speed of the ribosomes to use mRNA to make polypeptide chains (not functional proteins yet) by the process of translation (Lodish et al 86). This entire replication happens in about thirty minutes, and the speed of this duplication may be fine under normal conditions, but in a stressed environment mistakes can increase, which creates a stage for disaster. Stress in the cellular environment could be from increased demand, damaged organelles, depleted nutrients, fluctuations in pH, temperature changes, and many other variables; in other words, life is not easy for the cell, even under optimal conditions. Protein mistakes can lead to disease, mutations, and/or cancer. Increasing knowledge about the processes that create proteins, and how the cell can be manipulated to deal with these stresses and mistakes, will lead to safer treatments for people with less side effects. Ensuring that these proteins are created correctly and that ineffective proteins are dealt with properly upon discovery minimizes danger to the cell and prolongs the cell's life. Finding out how these mistakes manifest and finding ways to monitor and manipulate how the cell handles stresses will be invaluable to medical science.
One of the regulatory processes important for the creation of proteins is folding. The newly created polypeptides, secreted from the ribosomes may proceed to the endoplasmic reticulum (ER). ER's main function is to fold proteins into their functional structure before sending them out for use in the cell, like a factory creating finished goods out of raw materials. According to Dr. Zlatka Kostova, "Since proteins are translocated into the ER in an unfolded state, it is the primary function of this organelle to modify and fold the translocated proteins to acquire their biologically active conformation" (2309). Research has shown that the interactions between proteins and their processes is not unrelated, but instead the cellular processes are interdependent on each other's ebb and flow of nutrients and products within the cells' environment (Lodish et al 86). The ER takes the newly created polypeptides and adds certain substances, or moves the polypeptides to the Golgi apparatus, which packages and internally modifies the proteins for their final destination. ER alters an unfolded protein using chaperones, acts as a distributor for specific requirements of the cell, and determines where the protein should go next. Chaperones identify incorrectly constructed proteins and act as a monitor in the refolding process. Analogous to an inspector in a factory, chaperones are one of the defenses that the cell uses catch and incorrectly made proteins and degrade them if the proteins are deemed damaged or unnecessary. Utilizing this fundamental information about the nature in which proteins are synthesized the importance of the proteasome function becomes clearer because the environment of the cell can fluctuate and lead to conditions in which translational and protein-folding mistakes are amplified. As illustrated in Figure 1, an increase in mistakes can lead to mutations and/or death of the cell when the chaperones cannot cope with the excessive stress.
Chaperones are proteins that assist in folding new polypeptide chains and prevent aggregation. Chaperones are essential to protein quality control (PQC), helping to correctly fold new translated polypeptides at various locations in the cell. These chaperones are mostly found as heat shock proteins that act as quality control mechanisms for the factory environment of the cell (Wang et al 12). Proteins have charges and because of their hydrophobic interactions and tend to bunch up eliciting a response by the UPS, which will be discussed more in depth later. Large bundles of mis-folded proteins bunched together are known as aggregates, and one of the chaperones' main functions are to prevent the formation of these aggregates, which can impair the cell and cause autophagy (cell death) (Wang et al 11). Chaperones are within the ER, nucleus, and cytosol helping to correctly fold newly translated polypeptides. According to Dr. X. Wang, many of the proteins are imported to another area in the cell, which means the protein must translocate "across the membrane usually involving sequential unfolding and refolding, where many molecular chaperones play an essential role in this process" (12). The chaperone identifies the misfolded proteins, and acts as a supervisor by tagging "insufficient" proteins for their demise with an ubiquitin. Normally, the chaperone assists in ubiquinating these misfolded proteins before they become an aggregate, and the tagging of misfolded proteins with ubiquitin marks the proteins for destruction in a proteasome. This tagging of the proteins that have been targeted as insufficient links them to a polyubiquitin complex. The process of chaperones folding and re-folding the proteins in order to prevent aggregation of proteins is also previously illustrated in Fig. 1.
The inner matrix of the intercellular fluid, or cytosol, is the area in between the cellular components and serves as the medium for ions, nutrients, and proteins to move about in a microscopic, fluid-like environment. Continuing with the analogy of a cellular "factory," the proteasome regulates degradation within the cell and acts a garbage disposal along with the use of ubiquitin. Proteins created by the cell may last for just a short time, or they may last the life of the cell. Processes that the proteasome uses to regulate the cell require adenosine tri-phosphate (ATP) as a source of cellular energy, which will be discussed in depth later. This is a key point in understanding how cells modify their internal regularity. Protein regulation is crucial to the life and death of a cell as the cell goes throughout its cycles because if the proteasome is working correctly, it can prevent the onset of numerous diseases. When the proteasome is not functioning correctly, such as in the cases of certain cancers, the cell goes haywire and replicates non-stop. Some proteins may be created for a few moments while other proteins that are created are meant to last the entire life of the cell, and the proteasome must accurately regulate degradation in the cellular environment in order to maintain homeostasis (Su). Proteasomes remove both faulty and old proteins in order to prevent the build-up of garbage and "trash" within the cell.
This control of protein life span is referred to as protein degradation. Regulating degradation is imperative in maintaining the cell's structural integrity (Wang et al 2). Proteins are the foundational building blocks of all the cells in the body and they are essential in determining the cell's lifetime and nearly all of its processes (Sadava et al 327). The sheer number of proteasomes in an average cell emphasizes importance of this function to the survival of cells. The average mammalian cell contains about 30,000 proteasomes, and of these proteasomes there are varying forms that have been highly conserved throughout the course of evolution (Lodish et al 87). The culmination of the chaperones, PQC, and proteasomes symbiotically working together is the UPS.
The discovery of the ubiquitin-proteasome system has revolutionized the way scientists understand proteins at a molecular level. In 2004 the Nobel Prize Committee honored three men who assisted in the discovery of ubiquitin-mediated proteolysis. Aaron Ciechanover, Avram Hershko and Irwin Rose had been in collaboration with each other since the late 1970s working on a variety of experiments dealing with the recent discovery of ubiquitin ("Ubiquitin-mediated proteolysis"). Together, Ciechanover, Hershko, and Rose were trying to determine how the cell marks and internally degrades proteins after translation. As the name suggests, the ubiquitin-proteasome system, is quite literally ubiquitous all throughout the cell. The breakdown process that they wanted to understand is known as proteolysis. Proteolysis is the hydrolytic digestion of proteins or peptides into smaller amino acids by using enzymes known as proteases, which cleave protein at its peptide bonds through the enzymatic addition of water (Sadava et al 274). Ciechanover, Hershko, and Rose knew what proteolysis was, but they wanted to know how the cell targets proteins for this internal degradation and why this intracellular proteolysis required energy. Their eventual work to answer this problem led to the revolutionary discovery of the ubiquitin-mediated proteasome pathway, which shows that a large, complex system of enzymatic tags are used in the process of labeling proteins for degradation ("Ubiquitin-mediated proteolysis").
Understanding how ubiquitin works is a central idea in how the proteasome carries out its functions. According to the studies of Ciechanover and Hershko in the 1970s, the ubiquitin is a small seventy-six amino acid residue protein that becomes activated by enzymes (58). In the 1999 review, Schwartz and Ciechanover stated:
At present there are two predominant cellular systems for the degradation of cellular proteins: the vacuolar pathway (including lysosomes, endosomes, endoplasmic reticulum, etc) and the cytoplasmic ubiquitin-mediated pathway. In the late 1970s, Hershko & Ciechanover observed that abnormal cytoplasmic proteins were degraded at neutral pH in a process that required ATP. Additional studies revealed that multiple factors were required to support this property. (58)
Using this information, researchers in a controlled laboratory setting can alter the pH of the cell's environment to maintain or disrupt the delicate homeostatic range necessary for optimal proteasome activity and degradation of the cellular proteins. Ubiquitin is a small protein that is covalently linked to other cellular proteins identified for breakdown by the proteasome. Combining this information with the aforementioned material about proteolysis, one can begin to understand that the function of proteolysis is carried out by the proteases. By its own definition, the "proteasomes are large multi-subunit proteases that are found in the cytosol" (Bochtler et al 295). The property of cytoplasmic proteins becoming degraded in a neutral pH is important to the current research because cells must be kept in narrow range from 7.0pH-7.4pH, known simply as its "enzyme activity range" (Sadava et al 134). In the laboratory setting, researchers currently use a solution with a very specific and narrow range of pH called storage buffer. This buffer maintains the homeostasis of the cells, and if the cells are outside of this range then the cellular tissue lyses, or breaks down, and no further experimentation may be done on the proteasome or damaged cells.
To understand the function of the proteasome, the structure must be described. An evolutionary hint of the proteasome's importance is that the structure of the proteasome is similar in bacteria, archaea, and eukaryotes, and is highly conserved throughout the evolution of the cell (Volker et al 22). This information adds validity to the importance of researching within this field because if a cellular component has only slight variations within the cell throughout the course of its evolution, then that component's function must be essential to maintaining the cell's ability to survive and reproduce. Research has shown the importance of UPS in relation to its structure and physiology. After the cell recognizes an unfit protein, the cell targets this protein and sends it to the proteasome. The proteins that have been targeted as insufficient and linked to a polyubiquitin complex are given the so-called "kiss of death" as they are sent to the proteasome, which some have nicknamed the "cellular chamber of doom." It is inside of this catalytic chamber where the proteins suffer the morbid fate of the "death of a thousand cuts" that is carried out by enzymes using proteolysis to cleave the peptide bonds numerous times (Lodish et al 87). The name "chamber of doom" arises from the proteasome being a barrel-shaped protein complex made up of rings cylindrically-stacked that contain a catalytic core which internally breaks down the proteins. According Dr. Xuejun Wang:
The 26S proteasome which consists of a barrel shaped core particle (the 20S proteasome) and the activation complex (often the 19S proteasome) at one or both ends of the core particle. The cylindrical structure of the 20S proteasome is formed by an axial stack of 4 heptameric rings: 2 opposing identical Î² rings sandwiched by 2 identical outer Î± rings. Each Î± or Î² ring contains 7 unique subunits (Î±1 through Î±7, Î²1 through Î²7). (14)
Fig. 2 illustrates that the ends of the proteasome have "caps" that regulate the entry of ubiquinated proteins. There are several kinds of cap complexes, each with different activities, using energy from adenosine tri-phosphate (ATP), to remove the polyubiquitin complexes that have attached themselves to the protein that will be destroyed. Fig. 3 shows a more simplified version of the 20S and 26S proteasome and also illustrates the use of ATP to ubiquinate the protein. The catalytic core is the region of interest for many researchers because within its 1.7-nanometer diameter barrel, all of the protein is cleaved into short, reusable peptide products (87). Many processes that are essential for the continuation of the cell's life are carried out within this barrel-shaped structure, and continued research to expand the knowledge of this process could prove to be very beneficial.
The ubiquitin enzymes determine protein quality control (PQC). There are three types enzymes that activate ubiquitin, which floats freely around the cytosolic and nuclear regions of the cell and pair to proteins that will be marked for destruction, each of which may have hundreds to thousands variations or roles. The proteins necessary for PQC include the "ATP-dependent ubiquitin-activating enzyme (E1), which creates an activated ubiquitin that is transferred to one of a set of ubiquitin-conjugating (E2) enzymes. The E2 enzymes act in conjunction with accessory (E3) proteins, such as ubiquitin ligase" (Alberts et al 359). This three-step pathway leads to ubiquitination, and it is either immediately caught by a proteasome or it aggregates and is later degraded. The targeted protein is determined by the cell's PQC choices, and the "proteolytic pathway" is started upon detection of an "abnormal hydrophobic patch on a protein's surface" (358). This activated ubiquitin attaches itself to a lysine in the targeted protein, and along with the other ubiquitin chains, forms a "polyubiquitin complex," which then binds to the proteasome, and initiates the breakdown of the protein (Sadava et al 327). In contrast, deubiquitination enzymes recycle the ubiquitin to ensure that these enzymes are not wasted. These enzymes are also proteolytic, and if they were to escape the inner core of the proteasome they could potentially lead to the destruction of the proteasome and the entire cell. Like all of the functions in the cell, the deployment of ubiquitin must be monitored. Certain protein kinases that trigger responses in the cell can also regulate the proteasome. These protein kinases may be triggered at specific times when needed, and this process is known as signal transduction.
Signal transduction in the cell is the method that different tissues use to elicit physiological responses. Before describing protein kinase C (PKC), it is beneficial to know how protein kinases work. A well-known example of varying outcomes brought on by the same signal is the release of epinephrine, or adrenalin, into the bloodstream. Once in the bloodstream, epinephrine immediately triggers two vastly different responses in the body. In heart muscle cells, the epinephrine causes the pulse and heart rate to rapidly increase and stimulates blood to flow to the muscles; in contrast, in the stomach and digestive system the smooth muscle cell contractions are slowed down and blood leaves to go to the skeletal muscles (Sadava et al 339). In general, a protein kinase modifies other proteins in order to elicit a response, and it does this by changing the structure of the target protein by phosphorylation, which is the enzymatic addition of a phosphate. Upon the addition of this phosphate, the protein takes on an active form because ATP has been added to the protein and causes it to move to a new location and/or take continue in a chain of protein kinase phosphorylation down a signal transduction pathway. In the example of epinephrine, or molecular signal, elicited a different response, meaning that different cells can respond to a stimulus in a manner that is unique to the protein kinase pathway within the cell. In the case of UPS, PKC is speculated to have an effect on UPS function and its ability to keep the cell in equilibrium by coping with certain stresses.
The PKC pathway is one involving a membrane-bound enzyme that is influenced by the protein kinase pathway. The protein kinase cascade is defined as: "A series of reactions in response to a molecular signal, in which a series of protein kinases activates one another in sequence, amplifying the signal at each step" (Sadava et al 340). In the PKC, a hormone first attaches to a receptor bound on the membrane of a cell. Then the receptor activates G proteins that will cause enzymes on the inside of the cell to produce more secondary messengers that will open up a calcium channel to activate the PKC. PKC then continues down its own unique pathway to activate and phosphorylate enzymes and other proteins as well (341). These amplifications are very specific, and at different target proteins they can cause a unique response. It is speculated that PKC may have some sort of effect on how the ubiquitin-proteasome system carries out its functions.
Recent studies have shown that protein kinases are important in the function of signal transduction pathways in many cellular processes. The Serine/Tyrosine receptor kinase, PKC is involved in signal transduction pathways that "govern a wide range of physiological processes, including differentiation, proliferation, gene expression, membrane transport, and organization of cytoskeletal and extracellular matrix proteins" (Lu and Hunter 436). It is now known that the activation of protein kinases starts their downregulation of excess or useless proteins via the ubiquitin proteasome pathway. Although it still remains unclear as to how PKC is involved in the ubiquitination and degradation properties, any information about the relations between UPS and PKC will be found using well-established procedures in Dr. Wang's lab (Su). This lack of information shows the apparent need for increased research.
Before being able to obtain the proteins for a western blot, research must be done on the heart cells in vitro. This method has yet to be carried out, and is important for attaining new knowledge in this line of medical research which could lead to the discover of treatments for diseases. "In vitro" means that the experiment will be conducted without using a living system, such as a live mouse, which would be instead referred to as "in vivo". The cells used in the future experiments into this new field will be cultured in a medium that contains the nutrients necessary for their survival and experiments. The environment within the Petri dishes can be altered with drugs that either stimulate or inhibit the function of PKC. The function of the proteasome can then be examined by examining the proteasome substrates using GFPu/RFP, and there has been some research conducted in the X. Wang labs dealing with:
UPS proteolytic function in vivo, a proteasome function reporter system was established, in which a green fluorescent protein (GFP) was fused with degron CL1, and the modified GFP is referred to as GFPdgn or GFPu.66 GFPdgn/GFPu serves as a surrogate substrate for the UPS. In the absence of changes in synthesis, GFPdgn protein levels inversely reflect UPS proteolytic function. (Su and Wang 256)
This means that future research will show the effects based on the ration of GFPu/RFP in the case of PKC function alterations. Fig. 4 illustrates the effect of using skeletal and cardiac tissue from mice that have been genetically altered to show the function of the proteasome via GFP and is labeled with NTG meaning non-transgenic cells, or TG meaning transgenic cells.
This research using GFPu/RFP was recently discovered in 2008, and scientists can now observe the intricate processes within the cell. GFP is used by connecting it to a protein of interest, and after this genetic addition, researchers can then track the course of the protein to observe the effects of manipulating the proteins with drugs as illustrated previously in Fig. 4. According to a press release by the Nobel Prize committee, the importance of this discovery in 2008 can be used for:
Observing the building blocks of a cell: proteins, fatty acids, carbohydrates and other molecules are beyond the power of an ordinary microscope. And it is even more difficult to follow chemical processes within a cell, but it is at this detailed level that scientists must work. When researchers understand how cells start building new blood vessels, for example, they might be able to stop cancer tumors from acquiring a nourishing and oxygenating vessel system. This will prevent their growth ("The Nobel Prize in Chemistry 2008").
The research regarding PKC is important because once the effects of manipulation are known, further experiments can be conducted to develop treatments that may reverse the side effects if they are negative, or regulate them if they are positive. GFP is a bioluminescent protein, meaning that it omits light as energy, it absorbs ultraviolet light into a uniquely shaped structure known as a chromophore and then it radiates out the excess energy absorbed from UV light in the form of a fluorescent green glow ("The Nobel Prize in Chemistry 2008"). It is the ratio of GFPu/RFP that can be used as a reliable readout for proteasome function and it provides researchers with a visual of the proteasome substrates in the cells.
A prominent method that researchers in the X. Wang labs use to determine the effects of modifying proteins is called the western blot. Western blots are a common method within research labs used to detect proteins. Western blots use a process known as gel electrophoresis. In general, gel electrophoresis is the method used to separate charged particles in a gel based on their size and weight using an electric current to force these particles through the gel. After loading proteins into the wells of a polyacrylamide electrophoresis gel (PAGE), an electrical charge pulls the proteins down through the gel, which causes the proteins:
[to] migrate down through the gel matrix, creating lanes of protein bands. In native PAGE, migration occurs because most proteins carry a net negative charge at slightly basic pH. The higher the negative charge density (more charges per molecule mass), the faster the protein will migrate. At the same time, the frictional force of the gel matrix creates a sieving effect, retarding the movement of proteins according to their size (Electrophoresis Technical Handbook).
This basically means that the gel acts as a net to "catch" or impede the proteins on their path. Larger proteins separate slower and require a less dense gel, and the smaller proteins move quickly through the gel. In the outer wells of the gel, protein ladders that separate at specific "weights" allow researchers to find the size of the protein. Knowing this information, experiments can be designed that are specific to the size of the protein, and furthermore it allows a researcher to quantify the amount of a target protein in a sample during the later steps using analytical tools (Electrophoresis Technical Handbook).
This technique is also referred to as immunoblotting, due to its ability to label proteins by recognizing them as an antigen using antibodies. In order to label the protein that a researcher is trying to study, the protein needs to be recognized as an antigen by the antibodies used in blocking the proteins on the membrane. This "blocking" can be done using either an advanced blocking buffer or dry milk mixed into a phosphate buffered saline (PBS) solution. Blocking the membrane makes it so that the unnecessary proteins become bound to the free proteins in the milk/buffer solution, and at the same time the antibody can be added into this mixture at a determined ratio of antibody to solution. Through this labeling process a protein of interest is recognized as an antigen and binds to the antibody. With all of the other non-specific proteins being blocked and only the antibodies binding to specific sites, the amplification process is further amplified by using a secondary antibody western blot quantifies a specified protein in a cell lysate by a process of eliminating the other excess amounts of proteins. These other proteins that may interfere with the signal are "silenced" by using an antibody to select the protein of interest (Murphy et al 755). These methods to determine the amount of protein, and visualize the effects of stimulating or inhibiting the PKC signal transduction pathway will allow researchers to draw conclusions on PKC's role in UPS function.
In conclusion, medical science will gain valuable insight into the understanding of the proteasome by increasing the research into the unknown role of protein kinase C and how it may affect the homeostatic nature of the cell. The proteasome is a vital machine that the cell uses to deal with its internal protein regulation and degradation. Proteins made inside of the cell may turn on/off in order to regulate the many functions of the cell, and must be created in a fast paced environment that is usually under various forms of stress. These stresses that the cell encounters must be efficiently dealt with in order to keep the cell alive and prevent the onset of diseases caused by malformed proteins. Also the timed growth cycles of the cell must be maintained to replicate the cell during normal division, or upon fulfillment of the cells needs it may be terminated by apoptosis. Failures to be terminated are analogous to cancer where the cell replicates itself out of control and eventually leads to the death of the organism. The discovery of the proteasome has clarified how the cell regulates its proteins, which are important for nearly every aspect of the cells life, and has allowed researchers to manipulate the protein kinase cascades inside of the cell and discover the responses.
Although there is currently no information that links the UPS function and use of PKC, Drs. X. Wang and H. Su believe that this field will produce tangible results and elicit further studies. PKC is a signal transduction protein that currently has no research into its effects on the regulation of proteasome function and by increasing the research into this new line of molecular science researchers can experimentally examine how PKC affects the proteasomal activity. Even in the event that no new results are ascertained, this still would be considered beneficial because it increases researchers knowledge of the cells processes and it would simply redirect research to the next possible category. By studying the relation of PKC and its relation to UPS dysfunction, researchers can figure out how human diseases, such as neurodegeneration, heart failure, and various forms of cancer work and then develop treatments with minimal side effects.
I would like to acknowledge Drs. Huabo Su and Xuejun Wang at the Division of Basic Biomedical Sciences of the University of South Dakota for all of their help and contributions to this paper. I would also like to thank Sara Kniffen for her grammatical help and expertise from the USD Writing Center.
The following definitions are from Sadava's Eighth Edition Life: The Science of Biology.
Apoptosis: A series of genetically programmed events leading to cell death.
Bacteria: Unicellular organisms lacking a nucleus, possessing distinctive ribosomes and initiator tRNA, and generally containing peptidoglycan in the cell wall. Different bacterial groups are distinguished primarily on nucleotide sequence data.
Archaea: Some of the most primitive single celled organisms that live extreme conditions, and mostly located by heat vents in the ocean.
Eukaryotes: Organisms whose cells contain their genetic material inside a nucleus. Includes all life other than the viruses, archaea, and bacteria.
Differentiation: Process whereby originally similar cells follow different developmental pathways. The actual expression of determination.
Polypeptide: A large molecule made up of many amino acids joined by peptide linkages. Large polypeptides are called proteins.
Nucleic Acids: A long-chain alternating polymer of deoxyribose or ribose and phosphate groups, with nitrogenous bases-adenine, thymine, uracil, guanine, or cytosine (A, T, U, G, or C)-as side chains. DNA and RNA are nucleic acids.
Adenosine triphosphate: (ATP) An energy-storage compound containing adenine, ribose, and three phosphate groups. When it is formed from ADP, useful energy is stored; when it is broken down (to ADP or AMP), energy is released to drive endergonic reactions.