Discovery Of Ubiquitin Mediated Protein Degradation Biology Essay

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Prior to Schoenheimers research in 1942 it was believed that proteins were in a static state. Schoenheimer challenged this idea by labelling amino acids in proteins using stable isotopes such as 15N. His research found that proteins ingested were constantly being synthesised, degraded and interchanged in a steady state. [1] This triggered a huge interest and research area into how proteins are degraded which led to the discovery of the Lysosome by Christian de Duve in the mid 1950s. Hydrolytic enzymes function at an optimally acidic pH within the lysosome and are isolated from its substrates by a membrane. This provides controlled degradation of proteins. Autophagy explained how proteins are translocated into the lysosome. However, lysosomes could not give a satisfactory justification for varying protein half lives and sensitivities to lysosomal inhibitors at optimal pH. Most importantly, findings by Simpson in 1953 showed that generally in cells energy is required for the degradation of some proteins. He suggested that there must be two breakdown pathways, one of which is energy requiring. [2] The exergonic pathway of lysosomal protein degradation could not account for the use of metabolic energy. [3] Why energy was required for proteolysis triggered huge amounts of research into an energy dependent, protein specific and non lysosomal protein degradation pathway.

Energy is required for proteolysis in many cells. In 1964 Rabinovitz and Fisher investigated protein degradation in Reticulocytes (cells lacking lysosomes). Observations showed that Reticulocytes were able to degrade abnormal analogue containing Haemoglobin using energy. [4] This supported Simpson's ideas about an energy dependent non lysosomal pathway. And thus, ruled out speculations of lysosomes requiring energy in an indirect manner such as transporting proteins or for the proton pump to maintain an optimum pH in the lysosomal lumen.

In 1973 Etlinger and Goldberg further investigated non lysosomal protein degradation in reticulocyte cells. Reticulocytes were incubated in the presence of 2,4-dinitrophenol and absence of glucose. The results in figure 1 show that degradation of the abnormal ClAbu containing Haemoglobin was reduced under these conditions compared to when the cells were incubated with glucose and no 2,4-dinitrophenol.

Figure - The effect of metabolic energy on the degradation of abnormal ClAbu proteins in reticulocyte cells. Protein % breakdown was measured in a) presence of glucose b) absence of glucose and presence of the uncoupler 2,4-dinitrophenol (0.1nM) .Etlinger, Goldberg. 1977. A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Vol 74:55

In the absence of glucose, glycolysis and substrate level phosphorylation yielding ATP could not occur. [5] . 2,4-dinitrophenol is an uncoupler and dissipates the pH gradient in the electron transport chain inhibiting oxidative phosphorylation and thus ATP. The results strongly suggested that metabolic energy in the form of ATP caused a huge impact on the level of protein degradation. [6] 

Etlinger and Goldberg carried out various experiments to confirm ATP activates protein degradation. A cell free extract was created to simulate ATP dependent proteolysis in intact mammalian cells. Degradation of ClAbu containing protein was measured in the presence and absence of ATP at concentrations of 0.1-1mM. Their results (shown in figure 2) showed that ATP stimulated the degradation of abnormal ClAbu containing proteins compared to when ATP was absent.

Figure The effect of ATP on the degradation of abnormal ClAbu proteins. Protein % breakdown was measured in the presence and absence of ATP (1mM). Etlinger, Goldberg. 1977. A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Vol 74:56

The dependence of ATP on the degradation of these proteins was also investigated using ATP, ADP, AMP, cAMP and other ATP analogues. The stimulatory effects on protein degradation of these decreased as the potential energy from removing the terminal phosphate decreased from ATP to ADP. Both AMP and cAMP affected the degradation of proteins by a negligible amount compared to ATP and ADP as shown on the table of results in figure 3. The results evidently show that the cleavage of the terminal high energy phosphate provided the potential energy to stimulate protein degradation in an unknown mechanism. As this energy is highest in ATP it made sense that ATP stimulated degradation the most" [7] 8

Figure . The % stimulation of protein degradation caused by different 1mM Nucleotides compared to when no nucleotides are used. Etlinger, Goldberg. 1977. A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Vol 74:56

Further evidence of a non proteolytic pathway rose from measuring protein degradation in reticulocytes with ATP at varying pH levels. The optimal degradation observed was pH 7.8 (as shown in figure 3). This optimum pH is considerably higher than the acid optima characteristic of most lysosomal proteases. Their results completely and thoroughly ruled out any possibility degradation of proteins by any other mechanism other than ATP dependent proteolysis.7

Figure . Optimum pH for protein degradation in reticulocyte cells. In the presence and absence of ATP (1mM) the % breakdown of ClAbu containing proteins were measured at different pH concentrations. Etlinger, Goldberg. 1977. A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Vol 74:55

1980, Ciechanover .The discovery of a cell free system in which proteins are degraded using ATP stimulated research into the identification of a biochemical mechanism. To understand the ATP dependent proteolytic mechanism in reticulocytes, the underlying components were investigated by Ciechanover, Hershko an co. They fractionated the reticulocytes cell extract over diethylaminoethyl cellulose, removing haemoglobin and resulting with "Fraction I" and a salt eluate "Fraction II". The team observed that degradation of abnormal proteins only occurred upon combining the two fractions. [9] 

They then purified the fractions to isolate the active components in each. In fraction I, a heat stable, active component was isolated and named ATP-dependent Proteolysis Factor 1 (APF1) [10] . In fraction II a larger polypeptide which was stabilized by ATP was named APF2.

Now that APF1 was identified, its precise role in protein degradation was investigated. APF1 was labelled using radioactive isotopes and incubated in the presence of ATP and fraction II. The 125I-labeled APF1 showed an increased molecular weight on the SDS page. Upon removal of ATP the molecular weight of 125I-labeled APF1 decreased. [11] . They concluded that APF1 could reversibly and covalently bind to various proteins when fraction II and ATP were present but had no proteolytic activity on its own.

According to its molecular weight of 8.5kDa, characteristics and primary sequence analysis APF1 was identified as ubiquitin. [12] Ubiquitin was known to conjugate covalently to histones H2A and H2B in nucleosome by a high energy isopeptide bond requiring ATP [13] Due to APF1 protein conjugates resistance to sodium hydroxide and hydroxylamine, strongly suggested that, like ubiquitin and histones, APF1 binds to proteins covalently via an isopeptide bond.

Hershko et al 1980 Investigated the reversible reaction of APF1: protein conjugates upon the removal of ATP. They demonstrated the presence of deubiquinating enzymes (APF1 protein amide synthase) that reversed conjugation to liberate ubiquitin for another round of conjugation. The sequence of events were expressed as; covalent attachment of ubiquitin targets proteins for degradation, degradation of the target protein occurs ( by the 26S proteosome, unknown to them ) and release of ubiquitin for another cycle.

To identify the enzymes involved in the scheme, Ciechanover and Hershko isolated three enzymes These were identified as E1 (ubiquitin activating enzyme), E2 (ubiquitin carrier protein) and E3 (ubiquitin protein ligase). [14] .

They found that using ATP, ubiquitin was activated by forming a thioester link to a cysteine residue in E1 using its c terminal carboxylate. However, previous observations noted that binding of E2 to ubiquitin required E1 and ATP. This suggested that E1 transfers the ubiquitin to a cysteine residue on E2 and E2 transfers ubiquitin to the substrate. They also discovered that E3 specifically binds to substrates. Like antibodies, there are thousands of E3's which are specific to different proteins. This explained the varying half life's of proteins.

By 1983 the full system was understood and is still being used today ( figure 6). [15] The ubiquitin ligase (E2/E3 complex) left behind catalyses the formation of the isopeptide bond between ubiquitin and the target protein. The degradation signal on the target protein is recognised by E3. The target protein binds to ubiquitin ligase and a ubiquitin chain is added to the target protein.

A protease which identified the ubiquinated proteins required to be degraded was still undiscovered. Tanaka and her team identified another ATP requiring step in reticulocyte cells after the conjugation of Ubiquitin to the target protein [16] This suggested that the degradation of the target protein also required ATP. Hough and his team then discovered the 26s proteosome [17] made up of a 19s proteosome which regulates the entry of proteins by recognizing polyubiquinated proteins to be catalyzed and a 20s proteosome which has the catalytic activity [18] . This was proved correctly in 1990 by Hoffman and colleagues who mixed the 19s proteosome with the 20sproteosome to create the active 26s proteosome. The 26s proteosome degrades the polyubiquinated proteins into smaller peptides and releases ubiquitin [19] 

For a protein to be degraded it must be polyubiquinated. Once an ubiquitin molecule is attached to the target protein, another E1 molecule bound to ubiquitin transfers ubiquitin monomers to the target protein forming a polyubiquinated chain. As soon as 4 or more ubiquitin molecules are attached degradation becomes very rapid.


The discovery of ubiquitin mediated protein degradation was a five decade battle against logical and accepted protein degradation theories. One of the most powerful observations contradicting lysosomal degradation was the requirement of ATP in mammalian reticulocyte cells. This was the pivotal point leading research towards ATP dependent proteolysis. From 1977 to the Nobel prize in 2004 a simple observation caused a huge revolution in the protein degradation field.


1971 - Hershko and Tomkins suggest ATP participates as an energy source in enzyme degradation


1975- Goldstein et al Ubiquitin is isolated from bovine Thymus and later found in many tissues


1977- Etlinger and Goldberg - using rabbit reticulocytes degrading of abnormal properties without Lysosomes in an ATP dependent way

UBIQUITIN - E1, E2 and E3

1978- Ciechanover et al reticulocytes has Hb removed - spar solution separates into 2 fractions - individually inactive - when recombines ATD dependent proteolysis occurs

1979-Hershko, ciechanover and Rose discover the second fraction contains E3 and E1 enzymes.

1980- Ciechanoveer at al APF1 (ubiquitin) binds to covalently to a number of proteins in the lysate.

1980 ( Hershko et al) lysozyme, aa-lactalbumin and globin discovered iin APF1. Therefore, multiple polypeptides can be conjugated to the same substrate molecule.

1980- Wilkinson, Urban and HaasAPF-1 is recognised as ubiquitin - everywhere and therefore used in lots of cells. ATP is required due to its specificity and control.

1981-1983 Ciechanover, Hershko and Rose isolated the activities of E1 E2 and E3.

1981 Ciechanover et al -ubiquitin activating activity E1 is discovered.

Haas et al 1981: Ciechanover er al 1982- covalent affinity chromatograohy purifies E2 and E3.

Hershko et al 1983 -identification of subunits, ubiquiting-conjugatin enxyme E2, ubiquitin protein ligase E3 , ubiquitin activanting enzyme E1.


2002( ALberts et al ) Cells dont pass quality control adnd therefore ar sent for degradation