The Nobel Prize in Chemistry 2005

Autobiography

Richard R. Schrock

Ubiquitin, Prize Material

The Nobel Prize for Chemistry this year was awarded to three scientists who carried out pioneering research on ubiquitin, a eukaryotic protein that is so widespread from yeast to man that it gained its name as a derivation of the word “ubiquitous”. Aaron Ciechanover and Avram Hershko from the Israel Institute of Technology, and Irwin Rose from the University of California, pioneered research that unravelled the part ubiquitin plays in the degradation of proteins. Ubiquitin is now known to be involved in several other cellular processes as well, including quality control of nascent proteins, membrane trafficking, cell signalling, cell cycle control, X chromosome inactivation and the maintenance of chromosome structure, to name but a few. How can a tiny, 76-amino acid protein have so many varied roles? Ubiquitin (UB) acts through its attachment to other proteins (ubiquitinylation), and these protein modifications can alter the function or location of the protein, or target it for destruction. The C-terminus of UB extends as a 4-residue tail (Leu-Arg-Gly-Gly), where the terminal glycine residue can form an isopeptide bond with the amino group of a lysine side chain in a target protein. Alternatively, it can make an isopeptide bond with a lysine in another copy of UB to form a UB chain that ultimately attaches itself to a target protein. In general, the number and placement of UB molecules added to a protein helps to determine its future: a single copy of UB acts to modify a protein’s function, while multiple copies of UB will either modify a protein’s function or target it for degradation by the 26S proteasome, depending upon the position of the UB subunits. Cunningly, several pathogenic bacteria have developed the ability to tap into eukaryotic UB-mediated processes to manipulate their hosts. In particular, the host immune response can be suppressed through the inhibition of the NFkB signalling system. The transcription factor NFkB is responsible for activating genes involved in the immune response, but is inhibited by its association with the inhibitor IkB. The immune response is triggered by the ubiquitinylation of IkB to target it for degradation, which releases NFkB so it can enter the nucleus and transcribe genes required to mount an immune reaction. Certain bacteria are able to interfere with this system by either preventing the ubiquitinylation of IkB (as is found with non-pathogenic enteric bacteria that form part of the intestinal microflora), or by cleaving ubiquitin from IkB (as with the ubiquitin-like cysteine proteases found in several animal and plant pathogens) – either way, IkB evades degradation and suppresses the immune response by inhibiting NFkB.

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 Berne, a northeast Indiana farming community proud of its Swiss heritage. Life was not easy for my parents, Noah J. Schrock, the second of six children, and Martha A. Habegger, the second of ten children. They married in 1933 during the Depression. My oldest brother, Luther, was born in 1934, Theodore in 1939. A few months after I appeared in 1945, the family moved to Decatur, about 13 miles north of Berne, where we lived until the summer after my fifth birthday. My most lasting memory of our first home is its proximity to the city swimming pool where I spent many happy summer days.

We moved into an old house on the west side of South 13th Street in 1950. The house required a good deal of work, but my father, who had been a carpenter for fifteen years, accomplished the renovation over a period of several years. The house was located on what seemed to me to be an enormous plot of land (one acre); the backyard took forever to mow on a steamy summer day and the vegetable garden produced quantities of corn, strawberries, melons, tomatoes, and raspberries. A large vacant lot extended south toward a small railroad that dove beneath the highway on its way east to a mill where tiles were made from the clay dug on the west side of the road. The process of mining the clay created ponds, which I explored along with the surrounding woods at length. In the summer I would fish, catch snakes and frogs, and build simple huts in the woods, which probably were the first indications of my love for designing and building, and for the outdoors. In the winter I learned how to ice skate and often (so it seemed) would come close to actually freezing toes and fingers. We never had much money, but the house was comfortable (except during the first, and especially cold, winter) and the food (fresh in the summer, canned in the winter) was plentiful for the five of us.

The Schrock family in 1946.

My father built a woodworking shop in the two car garage at the rear of the house where I also spent much time discovering, among other things, that it is not easy to drive nails into maple. I was not allowed to operate power tools at a young age, but my father introduced me to several as I became older, except his prize 1941 Delta table saw, which I eventually inherited when he died of leukemia in 1980 at age 69. He was a patient teacher even though I did not fully appreciate the difference between a chisel and a screwdriver or clear pine and a prized piece of birds eye maple when I was young. I learned many things by trial and error but nevertheless grew to appreciate what one can do with wood and the right tools used the right way. I maintain that interest to this day. Like most young Hoosier men at that time, I played on the school basketball, baseball, and football teams. Although I did not continue to play organized sports beyond age ten or eleven, sports provided me with a respect and need for physical exercise that I still have.

My curiosity and love of building things played into the hand of my older brother Theodore who presented me with the proverbial chemistry set on my eighth birthday. He was thirteen at the time and beginning to appreciate the beauties of science. His interest in chemistry continued through his college years but he eventually studied medicine instead and became a highly successful surgeon. I was hooked. I created a small laboratory at the end of a storage area for canned goods and used my budding woodworking skills to build shelves for the ever expanding collection of test tubes, beakers, and flasks. I obtained most of my equipment through a mail order supply house with money earned from an early morning paper route. I carried out simple experiments (combining acids and bases to make salts, making pleasant smelling esters, etc.) following the directions in chemistry laboratory texts handed down to me. When I reached the age of thirteen, Harry Dailey, the high school chemistry teacher, stoked my interest in chemistry with more textbooks and discarded equipment. I thought all equipment wonderful to behold. As I became aware of the power of burning natural gas, the lowly alcohol burner was replaced by the common Bunsen burner, and the Bunsen burner ultimately by a high tech broad-headed model capable of putting out a good deal of heat, enough to melt metals in a porcelain crucible and even sodium chloride.

After we moved to a house on Jackson Street in 1958, my laboratory grew in size, diversity, and complexity. I now had at my disposal relatively sophisticated and, if misused, dangerous substances in a small room in the basement. I was often interested in testing recipes for mixtures of oxidizing agents and oxidizeable materials as well as nitrating common household substances. Thankfully, there were no serious mishaps, although my mother tells stories that belie that statement, including one in which the local fire department was called to our home; fortunately only a small rug was burning, not the house. At least I also was quick to think and act.

My father went to San Diego, California, in the fall of 1958 to work in the construction industry with his brother, Clarence, and to explore the feasibility of moving west. In 1959 my mother and I joined him. She drove and I navigated cross country with my laboratory carefully packed in the trunk of the car. By the time I finished Mission Bay High School in 1963, both my parents had also fulfilled their dreams of finishing high school, something they had not been allowed to do in their youth. My interest in chemistry expanded in San Diego. I found a laboratory supply house where I could buy classic equipment (a 250 mL retort was coveted and purchased) and a drugstore where I could buy basic chemicals with some adult help of course. I discovered many wonderful things such as how to make bromine from KBr and sulfuric acid, how to make sodium (and chlorine) by electrolizing molten sodium chloride, and how to analyze for metals by making sulfides with brilliant and characteristic colors. I took up surfing and skin diving, and continued my interest in woodworking by designing, making, and selling fins for surfboards. I entered a regional science fair with a project that concerned osmotic processes in sea urchin eggs and managed to win a prize for it. I collected the sea urchins at low tide and harvested the eggs myself. That, and dissection of a sheep's brain (which I greatly enjoyed) in physiological psychology later in college, was the closest I would come to following my brother into medicine.

I always assumed I would attend college and study chemistry. The only financially viable option was the University of California. I was accepted at Berkeley but chose to attend Riverside, a relatively new campus about 90 miles north of San Diego, because I thought that a smaller school might allow me to do more independent research earlier in my career. That proved to be the case. After the first exam in my first chemistry course at UCR, I was approached by Professor James Pitts who asked if I wanted a summer job. I agreed and began research in what broadly could be called atmospheric chemistry, a hot topic in the smog ridden Los Angeles basin and surrounding area at that time. In actuality, I spent my time learning to blow glass and construct vacuum lines, and to measure low concentrations of photolysis products using a temperamental, delicate, almost impossible to align, multi-pass Perkin-Elmer IR machine connected to a vacuum line. (Fourier Transform machines were not yet known.) A paper entitled "The Detection of Ethylketen and enol-Crotonaldehyde in the Vapour-phase Photolysis of trans-Crotonaldehyde" reported some of my work in 1968 after I had moved on to graduate school. I also worked "up the hill" with Dr. E.A. (Ed) Schuck and in 1966 found my name on a paper entitled "Rate Constant Ratios During Nitrogen Dioxide Photolysis." I learned many things, scientific and otherwise, that need not be detailed here. One that I might mention was the joy (and sometimes discomfort) of hiking in the Sierra Nevada Mountains. I still enjoy mountain hiking, although the frequency has decreased considerably. I capitalized on my knowledge of IR spectroscopy during a summer of research at Dow in Midland, Michigan, where my oldest brother Luther was an engineer.

Research as an undergraduate at the University of California at Riverside in the group of James N. Pitts in the mid sixties.

At Riverside I was influenced by a talented and enthusiastic teacher in physical chemistry named Jerry Bell. Jerry decided that I had enough ability to attend Harvard University for graduate study where he had received his Ph.D. I liked the idea, applied, and was accepted. I celebrated by listening to Rachmaninoff's second piano concerto played by Byron Janis, loudly, through a Fischer amplifier and large home built speakers, each with a volume approaching 12 cubic feet; an arts in western civilization course at UCR had boosted my interest in music that I had acquired in high school. During the last semester at UCR I took an inorganic chemistry course taught by Fred Hawthorne who appropriately spent a good deal of time discussing boron compounds. Although I enjoyed organic chemistry, the possibility of exploring the chemistry of all elements in the periodic table was fascinating to me. Yet for some reason I still regarded myself as a physical chemist.