According to Hershko, in order for them to find out what happens in the cell, they had to cut it open and make an extract. They then fraction the extract to separate the different parts and purify each enzyme component. Then characterized the mode of action of the purified components. The final step is the putting back the cell together and if it works, it means that one truly understands how the system works. They discovered that cells destroy unneeded protein in a phased process. The molecule, known as ubiquitin, attaches to a protein and accompanies it to a sac of powerful enzymes. This sac of enzymes break down the protein into the amino acids. The only proteins proven to be true are the ones that carry ubiquitin molecules and once the ubiquitin is done, it separates itself from the protein to be reused on another protein. Ubiquitin was unknown to other scientists and no one really knew what its job was. These scientists, Hershko, Ciechanover, and Rose figured out that ubiquitin tags to other protein that need to be destroyed and is taken to the proteasome which is known as the cellular trashcan. The protein is then signaled on its way for disassembly. If ubiquitin is under active, the cell could become hurtful (Avram Hershko – Lessons from the discovery of the ubiquitin system).In 1979, Hershko and Ciechanover used the observations of Etlinger and Goldberg which helped them in their discovery. Hershko and Ciechanover made the process split into two fractions (I and II) and had to recombine to form ATP dependent proteolysis. Fraction one contained a small, heat-stable protein they called APF-1. They found a high molecular weight fraction when they then examined fraction 2 (APF-2) and it was stabilized by ATP. It also needed to be reconstituted of the ATP-dependent proteolysis. APF-1 was ubiquitin and APF-2 was active protease. In their work, they tried to figure out whether an ATP-dependent association of APF-1 with other parts of the system. APF-1 with protein in fraction 2 was determined to be covalent. The bond was stable to NaOH. They concluded that conjugation was necessary for proteolysis. APF-1’s covalent attachment to cellular proteins explained why researchers had a hard time explaining the requirement for APF-1 in ATP dependent proteolysis. When fraction 2 was prepared from reticulocytes and didn’t use up the supplies of the ATP, most of APF-1 was shown in high molecular weight conjugates and it was found in fraction 2. The conjugates were taken apart in fraction 2, freeing APF-1. Because of this, enough of APF-1 was put in fraction 2 to support proteolysis. If one used up ATP, APF-1 was free before the chromatographic preparation of fraction 2 and APF-1 would have to be added back to gain the most amount of proteolysis. This experiment proved that APF-1 was covalently connected with multiple proteins in fraction 2 and the attachment was reversible. Although, it was not clear whether the modified proteins were enzymes of the system or that the substrates intended for degradation. To find out whether this covalent bond formation was similar to proteolysis, Hershko showed that authentic substrates of the system was changed a lot and the multiple molecules of APF-1 were stuck to molecules of substrate. Enzyme-catalyzed is what the conjugation was called, and for the first time it showed the activity of ubiquitin ligases. The ligases added more ubiquitin molecules to conjugates that already existed, even when there were excess free substrate. Proteomics say that there are hundreds of ligases of at least 2 different types. Therefore, it was likely that lots of ligases were occupied in fraction 2 and also explains why so many proteins were ubiquitinated. Almost 10 years later, another scientist named Chau proved that substrates for proteolysis were polyubiquitinated, which formed a chain K48 of one ubiquitin and the C terminus of the next. Hershko then observed that the conjugation was reversed during the removal of ATP by proving that enzyme-catalyst disassembly of conjugates and setting free of the ubiquitin and using it for another round of conjugation. This showed that specific amidases such as deubiquitinating enzymes caused the conjugation to reverse (Wilkinson, The discovery of ubiquitin-dependent proteolysis, PNAS). This work, done by Hershko, Ciechanover, and Rose, inspired a scientist named Keith D. Wilkinson. He worked with Rose in his laboratory. Once Wilkinson discovered about the amidases through the works of Hershko, he wrote an essay, specifically, deubiquitinating enzymes. He applied this work by purifying and cloning important regulatory enzymes of mammals, and another scientist named Miller, cloned the homologous protein from yeast. They then used the works of Varshavsky and his colleagues to learn that there are more than 80 of these enzymes and each of them are in at least 6 of the gene families which control important features ubiquitination (Wilkinson, The discovery of ubiquitin-dependent proteolysis, PNAS).The work that got the men Hershko, Ciechanover, and Rose a nobel prize changed and added some things to the study of chemistry. The first being the discovery of ubiquitin. Before people thought that protein degradation did not exist, but since they discovered the ubiquitin, they realized that it is possible. Since other scientists used the works of the men, it helped understand deubiquitinating enzymes and find out there are more than 80 enzymes (Wilkinson, The discovery of ubiquitin-dependent proteolysis, PNAS). Ciechanover was inspired to find out how proteins are eliminated because he believed people are in danger. He believed that since people are exposed to ultraviolet irradiation of the sun and people are breathing contaminated air. He was concerned that these things were very hazardous to the people. There are chemicals that change the proteins in human bodies and should therefore be removed (Aaron Ciechanover. Nobel Prize for the discovery of ubiquitin-mediated protein degradation).As a result of this discovery, other scientist and researchers are able to understand how diseases occur because the process of ubiquitin is uneffective. Irwin Rose and his team created a drug that treated blood cancer, known as myeloma, by killing the cancer cells with a pile of protein. The discovery also helped understand cell division and brain function. It also helped with disease, cancer, immune diseases, alzheimer’s, etc (Technion, Profs. Hershko & Ciechanover — First Israeli Nobel Scientists). According to Dr. De Bruin, with specific cancer types, cells produce too much protein which is higher than normal. If the process of protein degradation is stopped, the proteins pile up inside the cancer cell eventually causing the cell to die. Protoseams have six different active centers, and Dr. De Bruin figured out a way to study those centers all at the same time. He then created a molecules that can tie to the active centers and tried to stop their activity. For each active center he made an inhibitor and tried to study their roles in diseases like cancer. He blocked out two of the six active centers and was able to prove that it is possible to kill Leukemia cells (Leiden, Universiteit, Halting protein degradation may contribute to new cancer treatment, Science Daily). This work is very important because of all the new ways doctors have advanced their learning to treat illnesses. If Hershko, Ciechanover, and Rose hadn’t discovered protein degradation, people wouldn’t have known that proteins are removed from cells. Because scientists know that now, they figured out a way to remove cancer cells, treat Leukemia, and other types of diseases. This is especially very important to me because my grandma has cancer and she is using all her strength to fight it.