The Nobel Prize in Chemistry 2004

Aaron Ciechanover

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 in Haifa, a port city in the northern part of Israel, in October 1947, one month before Israel was recognized by the United Nations(UN) as an independent state. It took several additional months to establish the necessary institutions and for the British to leave, and on May 15th 1948, David Ben-Gurion, the founding father of the modern Jewish state and its first Prime Minister, made Israel a fact and declared its establishment as a democratic state and a home for every Jew in the world. The neighboring, but even more distant Arab countries, along with powerful Arab parties from within, did not accept the UN resolution and deliberately decided to alter it by force. A bloody and costly war erupted.
It lasted a year, and more than 1% of the population of the newly born and defenseless state sacrificed their lives on its defense. I assume that the first two years of my life (1947-1949) were extremely difficult for my parents, Bluma (nee Lubashevsky) and Yitzhak, who immigrated from Poland with their families as adolescents in the mid-1920s. Why did their families leave Poland - their "homeland" - their houses, working places, property, relatives and friends, and decided to make their new home in a place with a vague, if any, clear future, that was part of the British Empire? They were idealists who enthusiastically followed the call of the Zionist movement that was established at the turn of the century by Benjamin Ze'ev Herzel - the seer of the Jewish State - to settle the land and make it - after two thousand years in the Diaspora, since the destruction of the temple in Jerusalem - a home for the Jews.

Following the Jewish Congress in Basel (Switzerland) in 1896, Herzel declared: "In Basel I founded the Jewish State". At that time Israel was part of the Ottoman Empire and became in 1917 part of the British Empire. My parents came from religious families, and the move, I believe, had also religious roots: Jews, throughout their lives in the Diaspora, have not stopped dreaming of having their own country, with Jerusalem as its capital, a dream that was driven by a biblical decree and prophecy: "Thus saith the Lord GOD: Behold, I will take the children of Israel from among the nations, whither they are gone, and will gather them on every side, and bring them into their own land" (Ezekiel 37:21); "And they shall dwell in the land that I have given unto Jacob my servant, wherein your fathers dwelt; and they shall dwell therein, they, and their children, and their children's children, for ever" (Ezekiel 37:25); "And I will rejoice in Jerusalem, and joy in my people; and the voice of weeping shall be no more heard in her, nor the voice of crying" (Isaiah 65:19); "And they shall build houses, and inhabit them; and they shall plant vineyards, and eat the fruit of them" (Isaiah 65:21).

The question of timing was an important one, as despite centuries of continuous persecution and discrimination in Europe, the initial idea to establish a Jewish State had been the dream of a few. Only small groups of Jews settled in Israel during the 18th, 19th, and the beginning of the 20th century. It was only towards the end of the 19th century, with the ideas of Herzel and the moves that led to the Balfour declaration (the British Minister of Foreign Affairs, who declared in 1917 the recognition in the need for a Jewish homeland), that an active Zionist movement and institutions were established, resulting in the translation of the dream into reality. Yet, it took an enormous amount of courage and daring by these European Jews to materialize this dream and try to establish, with almost no resources or support, a homeland in a place they had dreamt of for two thousand years, but that was not theirs at the time. The process was clearly accelerated by the heavy clouds that then covered the skies of Europe and that ended with the Holocaust. Many members of my parents' families immigrated to Israel before the Holocaust, but those who remained in Poland were perished by the murderous German and their loyal Polish collaborators. The conversion of this movement into a State at that particular time (1947-1948) was no doubt the direct historical result of the holocaust, and symbolized the rise of the Jewish Nation from ash.

My father was a clerk in a law firm (later - along with my brother - he studied law and became a lawyer), and my mother was a housewife and English teacher.
My brother, Joseph (Yossi), who is 14 years older then me, was already on his national military compulsory service when I was 4 years old, the age from which I remember myself.

I grew up in Haifa and enjoyed the wonderful beaches and Mount Carmel that rolls into the Mediterranean Sea. From my early days at home I remember a strong encouragement to study.

My father worked hard to make sure we obtained the best possible education, and at the same time he was a member in the "Hagannah" (defense), one of the pre-state military organizations that fought the British for an independent Jewish State.

Working in a law firm in the Arab section of the city, he risked his life daily going to work during the pre independence war hostilities and then the war time.

My brother told me the family was waiting daily on the balcony to see him returning home safely. At home he used every free minute to delve into classic literature, Jewish religious law (Mishnah and Talmud) and modern law books. An important part of the education at home involved Judaism and Zionism. On the Jewish side we obtained a liberal modern orthodox education. We attended services in the synagogue every Saturday and during holidays, and celebrated at home all Jewish feasts.

Needless to say that my mother kept a kosher kitchen. It was extremely important for my parents to educate us as a new breed of proud Israeli Jews in their own independent country. My father inherited me with his love of Jewish studies and cultural life. To this very day, along with several physicians and scientists colleagues, I take regular lessons taught by a rabbinical scholar, on how the Jewish law views moral and ethical problems related to modern medicine and science. Jewish cantorial music reflecting prayers of Jews along many centuries has become my favorite music, and I avidly search for this vanishing vocal expression of Jewish culture in flea markets, used records stores, and auctions all over. Also, different Judaica artifacts decorate my study. In parallel, my parents made sure we should receive an excellent general education. My father spoke fluently several languages, Hebrew, Polish, Arabic, French, English, German and Yiddish, and wanted me to acquire his strong love for books: while our home was not a rich one, we had a huge library. My parents also loved classical music, so we had a great collection of 78 rpm, and later 33 rpm records. I remember that Bizet's Carmen occupied more than twenty, double-sided, RCA (His Master's Voice) 78 rpm bakelite records.

The apparently peaceful life of our family in Israel (although under the British Crown) during the years of the Holocaust in Europe (1939-1945) were overshadowed by the murder of their family members and of many families of their relatives and friends that did not escape Europe in time. For my parents, the establishment of the State of Israel as an independent and sovereign Jewish State was a direct historical result of the Holocaust in Europe and a clear statement of "Never Massadah shall fall again" (Massadah was one of the last strongholds of Jews during the Roman Empire. It fell into Roman hands after a long curfew during which all its defenders committed suicide in order not to fall as prisoners in Roman hands. While asiring for freedom, they lost their land and lives. They were not ready to live anywhere or under any circumstances, but as free people in their own land).

They left us with the idea that the Jewish State will not only protect us as a free people, but will allow us to develop our own unique culture in a more general national context, rather than as minorities scattered in different countries in the Diaspora.

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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.
This is my BrainyGoose:
United States, IL, Chicago, English, Italian, Genry, Male, 21-25, bodybulding, swiming.

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