ProteasomeUbiquitin-mediated Protein Degradation
The addition of a chain of multiple copies of ubiquitin (UB) targets a protein for destruction by the intracellular protease known as the 26S proteasome, a large complex that breaks down proteins to their constituent amino acids for reuse. The proteins targeted by this system are short-lived proteins, many of which are regulatory proteins, whose actions are controlled in part by rapid synthesis and degradation, much like an on/off switch; as such, the UB system itself is an important regulatory tool that controls the concentration of key signalling proteins. For example, many cell cycle regulatory proteins, such as cyclin, are controlled by UB-mediated proteolysis to allow a rapid transition between cell cycle stages, and to drive the direction of the cell cycle by preventing regression to an earlier stage. The selective UB-mediated degradation of proteins is also involved in the stress response, antigen processing, signal transduction, transcriptional regulation, DNA repair and apoptosis.
In addition, the 26S proteasome targets misfolded, damaged or mutant proteins with abnormal conformations that could be harmful to the cell. UB-dependent proteolysis provides the cell with a proofreading capacity for nascent polypeptide chains, whereby faulty polypeptides are targeted for destruction. Sequences that signal UB-mediated destruction can be buried in a hydrophobic core, which only becomes exposed after misfolding, providing a convenient way to distinguish misfolded proteins from functional ones - however, the presence of chaperones protects a polypeptide from degradation from the time it is synthesised until it is fully folded. Damaged proteins are also targeted. For example, hepatic cytochromes P450 are haemoproteins engaged in the oxidation of endo- and xenobiotics, during which they can become damaged by reactive intermediates; these damaged liver enzymes are rapidly removed by the UB-dependent proteolytic system.
It is important for a cell to be able to select specific proteins for degradation so as to avoid degrading proteins vital to the functioning of the cell, as well as to precisely control the delicate balance that exists between the proteins in a regulatory system, and to cope with the cell’s ever-changing protein requirements. The ubiquitin-mediated pathway achieves a high level of specificity, selecting only UB-tagged proteins to be destroyed. In addition, there exists a class of enzymes that function to remove UB from substrate proteins, thereby rescuing them from destruction by preventing indiscriminate degradation. Thus, for a protein to be degraded, it must not only have some type of UB-tagging signal, but also must escape the de-ubiquitinylation enzymes. The attachment of UB to a target protein requires the action of three enzymes, called E1 (UB-activating enzymes), E2 (UB-conjugating enzymes) and E3 (UB ligases), which work sequentially in a cascade:
Ubiquitin activation
E1 enzymes are responsible for activating UB, the first step in ubiquitinylation. The E1 enzyme hydrolyses ATP and adenylates the C-terminus of UB, and then forms a thioester bond between the C-terminus of UB and the active site cysteine of E1. To be fully active, E1 must non-covalently bind to and adenylate a second UB molecule. The E1 enzyme can then transfer the thioester-linked UB to the UB-conjugating enzyme, E2, in an ATP-dependent reaction.
Ubiquitin conjugation
UB is linked by another thioester bond to the active site cysteine of the E2 enzyme. There are several different E2 enzymes (>30 in humans), which are broadly grouped into four classes, all of which have a core catalytic domain, and some of which have short C- or N-terminal extensions that are involved in E2 localisation or in protein-protein interactions. The different E2 enzymes are able to interact with overlapping sets of E3 ligases.
Ubiquitin ligation
With the help of a third enzyme, E3 ligase, UB is transferred from the E2 enzyme to a lysine residue on a substrate protein, resulting in an isopeptide bond between the substrate lysine and the C-terminus of UB. UB ligation provides the key steps of substrate selection and UB transfer to the protein target, with the E3 ligases being responsible for substrate specificity and regulation of the ubiquitinylation process. Hundreds of putative E3 ligases have been identified, which bind to specific substrate sequences, or “degrons” (as they are targets for degradation), permitting the substrate specificity associated with this enzyme. There are at least four classes of E3 ligases: HECT-type (IPR000569), RING-type (IPR001841), PHD-type, and U-box containing (IPR003613). The E3 ligases are the only one of the 3 enzymes that is subjected to regulation, however balance in the UB system is also achieved through a set of de-ubiquitinylating isopeptidases that cleave UB off substrates.
Ubiquitin elongation
Additional UB molecules can be linked to the first one to form a poly-UB chain, which occurs through a particular type of E3 ligase sometimes referred to as a UB-elongation enzyme, or E4. There are seven lysine residues in UB that can be used to link UB molecules together, resulting in diverse structures. Poly-UB chains linked at different positions alters the destiny of the target protein to which it is added: Lys(11)-, Lys(29)- and Lys(48)-linked poly-UB chains target the protein to the proteasome for degradation, while Lys(6)- or Lys(63)-linked poly-UB chains (as well as mono-ubiquitinylation) signal reversible modifications in protein activity, location or trafficking. The length of the UB chain appears to be important as well, such as with Lys(48) poly-UB chains where its length influences its affinity for proteasomes. Therefore, E3 ligases provide the exquisite specificity in regards to which proteins should be targeted with UB, how many UB molecules are added to the target, and at what positions the poly-UB molecules are linked, thereby determining the future of the protein and the precise role it will play.
Proteasome
The 26S proteasome is a large (>60 subunits) complex with a 20S barrel-shaped proteolytic core consisting of alternating a and b subunits, and two 19S regulatory “caps” at either end (see diagram above). The 19S caps recognise, de-ubiquitinylate and unfold the target protein before it is pulled through the hollow core of the 20S catalytic centre, where it is dissembled into reusable amino acid components.
Disease
Inappropriate UB-mediated protein degradation has been implicated in a number of pathological conditions, especially neurodegenerative disorders that involve protein aggregation and inclusion body formation, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and ALS, where protein misfolding may play a role. Several Parkinson’s disease-causing mutations have been identified in genes encoding for UB-mediated degradation pathway proteins, such as the PARK2-encoded Parkin protein that causes autosomal recessive juvenile parkinsonism (AR-JP), and which appears to function as an E3 ligase. This degradation pathway is also implicated in certain forms of cancer as well.
We left my birthplace, Brooklyn, New York in 1939 when I was 13. I enjoyed the ethnic variety and the interesting students in my public school, P.S. 134. The kids in my neighborhood were only competitive in games although unfriendly gangs tended to define the limits of our neighborhood. The major extracurricular activities that I can remember were a Victory Garden on school grounds, our contribution to the war effort, and a favorite sport, handball, played between the walls of our apartment house.Mother, Ella Greenwald, was an American born into a family that included one sister and four brothers, all born in Hungary. Father, Harry Royze, had two brothers and a sister from the Odessa region of Russia. The Greenwalds and the Roses were secular Jews and the children more so although my younger brother and I spent some time in Hebrew school to please Grandfather Rose.
Due to my brother having rheumatic fever the family was advised to go to a high and dry climate, Spokane, Washington, where my mother's sister had a comfortable home that could accommodate us. This left my father behind tending his flooring business, an arrangement that I never understood and felt conflicted about. Father's visits were few and far between. The war was going on. Mother did secretarial work in the Navy Supply Depot in Spokane while we kids were making our way through the Spokane school system.
I worked during the summers at a local hospital, chiefly helping out in the psychiatric ward. In time I came to see myself following some career that involved solving medical problems. No one in my family had followed a career in research. Uncle Arthur G. was an excellent violinist and artist, and taught cabinet making at a trade school in Brooklyn. Uncle Dave R. would have become a lawyer had the economic depression not led him into the U.S. Internal Revenue Service. There was no one in my circle from whom I could expect to get advice.
Initially, I thought problems on how the brain works to be the most interesting. But it was necessary to be practical, and concentrate on less obscure matters when I entered Washington State College. Besides, there were no courses given in neurobiology. However, I was strongly influenced by Prof. Herbert Eastlick, who urged his zoology students to set high standards for themselves, and then proceeded to the University of Chicago after a brief period in the Navy. My PhD thesis problem was to determine if the DNA content of rat tissues increased if there was B12 in the diet. This problem was suggested by my adviser based on the observation that thymine could replace vitamin B12 in a lactobacillus. I analyzed the DNA of tissues of rats fed with diets that varied in B12. This project was doomed to failure when the genetic nature of DNA was revealed, and I found that the DNA content per cell of liver was independent of diet1.
PhD Work. I had to think of a new thesis project. Anxious to make up for lost time, I picked a problem out of my freshman biochemistry lecture notes. The Putnam/Evans group was interested in determining the origin of the nucleic acid components of bacteriophage synthesized in E. coli and Frank Putnam's lectures described experiments of Hammarsten, Reichard, and Saluste2 as background information. 15N-cytosine, the free base, had been found not to be incorporated into DNA although 15N-cytidine was incorporated into rat liver DNA. It was obvious for me to ask if there might be direct utilization of the whole of cytidine, ribose and all, in the biosynthesis of deoxycytidine. That would be a shock. I learned from Peter Reichard, during a 2004 meeting in Stockholm, that the export to Sweden of 14C-compounds was forbidden by the U.S. Atomic Energy Commission at that time, otherwise they certainly would have done the obvious follow-up experiment, using uniformly U-14C labeled cytidine themselves.
I made RNA from Euglena gracilis grown on 14CO2. I had to work out the determination of the independent specific activities of the sugars and bases which I did by treating the nucleosides with nucleoside phosphorylase and hypoxanthine to exchange for the base to be analyzed. Then by paper chromatography, using a medium containing borate to retard the migration of ribosides, I could also isolate deoxyinosine and cytosine. Although U-14C cytidine did not label the deoxyribose of E. coli DNA, I found the deoxycytidine of DNA of rat organs to be almost uniformly labeled. The 14C content was far in excess of the negligible radioactivity in the purine deoxynucleotides3. Therefore by both criteria it appeared certain that the 14C reached the deoxyribose directly from the cytidine. Reichard repeated and extended this experiment with U-14C uridine in 1957 with much the same result for the deoxycytidine and thymidine4.
It would have been reasonable for me to try to work out the enzymology of ribonucleotide reduction after graduating. Peter Reichard at Yale on a post-doc from Sweden, asked me about my intentions. But I was not anxious to take on a heroic problem at this early point in my career. I was interested in learning more about the principals of enzymology.
1950 at Chicago.
Stereochemistry at Chicago. Ogston's 1948 paper proposing, in effect, that the ability of an enzyme-substrate complex to distinguish between identical groups on a tetrahedral carbon was a consequence of the asymmetry of the complex5, was a matter of hot debate in chemistry/biochemistry circles at Chicago in 1950 where the enzyme was still a black box and the emphasis was on the chemistry of changes in the substrate. In particular the Ogston idea could justify the conclusion in the experiments of Myron Bender, done in Chemistry at University of Chicago, that the absence of back labeling of an ester in 18O-water during enzymatic hydrolysis could not rule out a tetrahedral intermediate. Bender had already shown that back labeling occurred during ester hydolysis in alkali6. In the case of the enzymatic reaction based on Ogston one would expect to lose all the 18O on stereospecific return of such an intermediate to the ester. These thoughts morphed into the positional isotope exchange idea in 1976.
1997 at Fox Chase.
Thus I became challenged to establish the absolute stereochemistry of enzymatic reactions and determine its mechanistic significance, if any. This did not seem such a formidable task, although it was not until 1963 that Kenneth Hanson and I solved the historically important problem of the prochirality of citric acid7, which was necessary for me to gain a proper perspective on the aconitate hydratase reaction.
Yale. In 1955, after post-doctorals at Western Reserve University with C. E. Carter and at New York University with Severo Ochoa, I was fortunate to be invited by Joseph Fruton to become an Instructor in Biochemistry at Yale University Medical School. The first year at Yale was notable for the following three developments. Not willing to spend the time it would take to get the Department's mass spectrometer working, I turned to the scintillation counter that was available in the medical school lab of Seymour Lipsky, an M.D. with a passion for exploring and exploiting new methods. One of the pioneer instruments to become available came from a small start-up company in New Haven that Lipsky had been encouraging, the Technical Instrument Company. Lipsky also had a sample of tritiated water which together with his counter got me started on experiments I wanted to do.
Fructose-6-P formation from glucose-6-P in H2O and D2O, as measured colorimetrically by the Roe test for ketoses.
A second important event of my first year at Yale was to learn from Mel Simpson of his paper showing an apparent ATP requirement for protein breakdown in a liver slice system. This observation required further study which I attempted on the side for the next twenty years.
But the crowning event of 1954-1955 was my proposal of marriage to Zelda Budenstein, a graduate student in the Department. Fortunately, I caught up with her before she graduated. Her mother, widowed since Zelda was age five, came to live with us. She was much loved and a great help with the four children that were in our onrushing future. She enabled Zelda to have a research career, often paralleling mine, which she continued until 1987 when she retired to devote full time to her peace and social interests.
Aldose-ketose isomerases. Probably the most interesting experiment of my nine years at Yale, l954-1963, was interesting from the way it developed and the confidence it gave me that I might be able to do research after all. I had been looking for evidence of proton transfer in enzymes that catalyzed aldose-ketose interconversions. We had been mistakenly unsuccessful in not finding the small amount of transfer that was later detected in the triose P isomerase reaction. Y.J. Topper had reported that glucose 6-P isomerase in D2O formed glucose-6-P (G6P) containing about one deuterium using crystallization of the barium salt as a G6P trap8 suggesting that there might be no proton transfer between reactants of this enzyme. The importance of showing some transfer would be that it would provide a clue to the catalytic process. Complete transfer would suggest a hydride transfer. Complete exchange would suggest a carbanion intermediate but would not implicate the enzyme as the base. However, the occurrence of both transfer and exchange would result if the abstracted proton were to exchange to some extent before a second proton transfer. Such a result would imply a single base mechanism. No such result had yet been reported. In unrelated experiments I observed a puzzling phenomenon with G6P isomerase. When G6P was used with isomerase in D2O the colorimetric analysis for fructose-6-P passed through a maximum before reaching a final value. This overshoot of equilibrium that occurred only in D2O, Figure 1, was very puzzling.
Professor Julian Sturtevant of the Chemistry Department sagely asked me if I was sure of my assumptions. I soon figured out that Topper's experiment might have been misleading. Perhaps his barium trap of G6P was not good enough. If there were both transfer and exchange in D2O then as the product returns to the enzyme there would be another opportunity for exchange until the product becomes fully exchanged. The fructose-P would go from a partly H- form initially, to an all D-form in the exchanged position at C-1. Now the only thing necessary for my strange result to make sense was to find an isotope effect in the color reaction for ketoses in acid. This was shown by finding the equal amounts of fructose-6-P at 20 hrs when assayed by a different method9.