The Nobel Prize in Chemistry 2004

Avram Hershko

Ubiquitin-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.

I was born on December 31, 1937, in Karcag, Hungary. Karcag is a small town of around 25,000 inhabitants, about 150 kilometers east of Budapest. It had a Jewish community of nearly one thousand people. My father, Moshe Hershko, was a schoolteacher in the Jewish elementary school in Karcag; most of the Jewish children in that town were his students. His former students from Hungary, and later from Israel, described him with admiration as an inspiring teacher and a role model educator. My mother Shoshana/Margit ("Manci") was an educated and musically gifted woman. She gave some English and piano lessons to children in Karcag. My older brother, Chaim, was born in 1936, less than two years before me. My mother wanted very much to have also a baby girl, but the times were at the eve of World War II, Hitler's screams could be frequently heard on the radio, my parents became apprehensive of the future and thus did not try to have more children. Still, my recollections of my early childhood are of very happy times, with loving and supporting parents, growing up in a nice house with a beautiful garden, created by my father who was also an amateur (but avid) gardener. A family picture from these times, with my parents, my brother and I as an infant, shows well the warmth of my family.

This early paradise was lost rapidly and brutally. World War II broke out, and soon Hungary joined in as an ally of Nazi Germany. In 1942, my father was taken by the Hungarian Army to serve as a forced laborer, in the company of other Jewish men. They were sent to the Russian front, where most of them perished. Luckily for my father, the Soviet Army advanced so rapidly after Stalingrad that he was captured by the Soviets before the Nazis could kill him. Then, he was used by the Soviets as a forced laborer. He was released only in 1946, so we did not know for four years whether or not he was alive.

In the spring of 1944, Hungary's dictator Horthy understood that Germany was loosing the war, and planned to desert. The Germans sensed this and quickly occupied Hungary. This was followed by the rapid extermination of much of the Jewish population of Hungary. In May-June 1944, most Jewish people were concentrated in ghettos and then transported to death camps in Poland. I was six years old at that time. We were in a ghetto at the outskirts of Karcag for a couple of weeks and then were transferred to a terribly crowded ghetto in Szolnok, which is a larger city in the same district. From there, Jews from the entire district were transported further on freight-trains. They were told that they were sent to work, but after the war we learned that most of the trains were headed for Auschwitz. By some random event, my family and I were put on one of the few trains that headed for Austria, where Jews were actually used for labor. This group included my mother with us two children, my paternal grandparents and my three aunts. In Austria we were in a small village near Vienna, where adults worked in the fields and in a factory. We were liberated by the Soviet Army on the spring of 1945. My maternal grandparents perished in the Holocaust, along with 360,000 Hungarian Jews and almost two-thirds of the Jewish people of Karcag. Following our reunion with my father in 1946, the family lived for three years in Budapest, where my father found job as a schoolteacher. The family emigrated to Israel in 1950.

My parents, my brother (middle, up) and I (middle, down) at around the end of 1938.

In Israel we settled in Jerusalem and I started a new and very different life. Of course, there were initial difficulties of being new immigrants. We had to learn a new language, Hebrew. This was not too difficult for children (I was less than 13 at that time), but it was more difficult for my parents. Still, my father studied Hebrew and soon started to work, again as a schoolteacher. (Later he taught in at a teachers' seminary and authored mathematics textbooks, which were very popular in Israel). As always, education of their children was my parents' highest priority. Although we were quite poor immigrants at that time, my brother and I were sent to an expensive private school in Jerusalem. I suspect that most of the salary of my father was spent on our tuition fees.

At school I was received well by the other children. These were times of massive immigration to Israel, so a new immigrant child with a Hungarian accent did not stand out too much (I am told that I still have some Hungarian accent, especially in English, though my Hungarian language is quite poor now). I was a good student, and learned easily different subjects, such as mathematics, physics, literature, history and even Talmud! That became a problem when I finished high school. I was interested in too many subjects, so it was difficult for me to decide how to continue. I chose to study medicine, probably by default, because my brother Chaim was already a medical student, so I could inherit his books for free! Chaim always wanted to be a physician, and he is now a very well-known hematologist and an authority on iron metabolism.

In 1956, I started to study at the Hebrew University - Hadassah Medical School in Jerusalem, which was the only medical school in Israel at that time (there are now four). In the basic science part of my medical studies, I fell in love with biochemistry. I studied biochemistry in three different courses: organic chemistry, basic biochemistry and a course called "physiological chemistry", which was medically oriented biochemistry. I was very fortunate to have outstanding teachers in all three courses. Organic chemistry was taught by Yeshayahu Leibowitz, a legendary person in Israel, a highly original thinker whose knowledge encompassed philosophy, political science, the Bible, Talmud, medicine, chemistry and more. He was probably my best teacher, it was an intellectual feast to listen to him. Leibowitz loved biochemistry, and he sneaked biochemistry into his lectures on organic chemistry whenever he could, which was often. Basic biochemistry was taught by Shlomo Hestrin, also an inspiring teacher who had a special talent of transferring his enthusiasm for science to the students. Physiological chemistry was taught by Ernst Wertheimer, a professor of German Jewish origin whom we had some difficulty to understand because of his heavy German accent, but who had an excellent perspective of integration of metabolism at the level of the total body and of physiological contexts of biochemistry. Another part of the same course was taught by Jacob Mager. Mager was an outstanding biochemist and a man of encyclopedic knowledge. However, he was very shy and quite a bad classroom teacher (though an excellent teacher in the laboratory, as I learned later). Most of his lectures were delivered while he was writing whole metabolic pathways on the blackboard, without any notes, with his face to the blackboard and his back directed to the class. Still, I was so much impressed by the depth and breadth of his knowledge of biochemistry that I decided to ask Mager to do some research in his laboratory.

Jacob Mager.

I started to work in Mager's laboratory in 1960. At that time, there was no formal M.D.-Ph.D. program at the Hebrew University, but it was possible to do a year of research between the preclinical and the clinical years of medical studies. I did that, and although I completed medical studies later on, I already knew by the end of that year that I was going to do research, rather than clinical practice. I was very fortunate to have had Jacob Mager as my mentor and tutor of biochemical research. He was a scientist with incredible scope of interests and knowledge. He was interested in every subject in biomedicine, he knew almost everything about every subject and he worked simultaneously on 3-4 completely different research projects. This undoubtedly caused fragmentation of his contributions to science, but provided his students with a broad experience in different areas of biochemistry in a single, relatively small laboratory. In a period of a few years I worked with Mager on subjects as different as the effects of polyamines on protein synthesis in vitro, glucose-6-hosphate dehydrogenase deficiency and a variety of aspects of purine nucleotide metabolism, including enzymology and regulation. During this time, I also finished my medical studies, did my military service as a physician (1965-1967) and then returned for two more years to Mager's laboratory to finish my Ph.D. thesis (1967-69). I received not just a broad view of biochemistry from Mager, but also a very solid base. He was a very rigorous experimentalist, every experiment had to be done with all possible positive and negative controls, all experiments were carried out in the duplicate, and every significant new finding had to be repeated several times to make it sufficiently credible. I owe a lot to Jacob Mager for a strong background of rigorous biochemistry.

Judy and Avram Hershko in 1977.

I met Judith (n?e Leibowitz) in 1963, and we married at the end of the same year. Judy was born and raised in Switzerland. After her studies in biology, she decided to spend a year in Israel. During this year, she worked in the hematology laboratory of the Hadassah hospital in Jerusalem. One day, I walked over to the hematology laboratory to get a blood sample that I needed for my research, and we literarily bumped into each other. This collision caused her to stay in Israel for more than one year, and now we are married for over 41 years. We have three sons: Dan (1964), Yair (1968) and Oded (1975). Dan is a surgeon, Yair is a computer engineer and Oded is a medical student. We have now six grandchildren: Maya (1994), Lee (1997), Roni (1998), Ela (2000), Ori (2002) and Shahar (2004). Needless to say, both Judy and I are crazy about all our grandchildren. During all our years together, I got tremendous support from Judy. Although she came from one of the world's most peaceful countries to one of the least, and from a very comfortable and pampering environment to quite primitive surroundings, she stood her ground with a lot of energy, courage and cheerful optimism. She always took care of all my possible needs, as well as the needs of our children and grandchildren. Judy is not only a very beautiful woman, but she also radiates a lot of caring, love and compassion. In addition to providing so much support at home, she also helped me a lot in the laboratory over a period of more than 15 years. The ubiquitin system was helped by Judy in more than one way.

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Genry/Male/21-25. Lives in United States/IL/Chicago, speaks English and Italian. Eye color is brown. I am muscular. I am also passive. My interests are bodybulding/swiming.
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United States, IL, Chicago, English, Italian, Genry, Male, 21-25, bodybulding, swiming.

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