The Nobel Prize in Chemistry 2004
The return to Israel – independent research career
After three years at M.I.T. (1981-1984), it was time to seek for an independent academic position. After many deliberations and despite attractive offers and a strong temptation to stay in the US, I decided to return home, to Israel. With the help of Avram, I obtained an independent academic position in the Department of Biochemistry at the Faculty of Medicine of the Technion (where I graduated), and returned home towards the end of 1984, after a productive post-doctoral period. Importantly, I already had a research subject I wanted to pursue, the effect of RNase on ubiquitin-mediated proteolysis.
The years that followed the post-doctoral fellowship (1984-present) have been extremely rewarding. I was happy to return to Israel, to my family and friends, to a place I felt I belong. I established my own independent research group and laboratory, obtained extramural competitive funding, and continued my research on the ubiquitin system. I have been lucky to have, along the years, a group of extremely talented graduate students and post-doctoral fellows. In our first series of studies we elucidated the role of tRNA in the proteolytic process, a subject I discovered as a graduate student and continued to study independently while at the M.I.T. (see above). Along with one of my first graduate students, Sarah Ferber, we demonstrated that proteins with acidic N-termini, Asp or Glu, undergo arginylation at the N-terminus, converting the acidic, negatively charged residue at this site to a positively charged residue. The reaction is catalyzed by Arg tRNA-protein transferase, a known protein with an hitherto unknown function. The enzyme uses charged tRNAArg as a source of activated Arg. Therefore, digestion of the cell extract RNA with RNase A inhibits this reaction. This finding explained the selectivity of the RNase effect to BSA and not to lysozyme: BSA has an Asp residue in the N-terminus, while lysozyme has lysine in this position. Interestingly, the ligase involved in BSA ubiquitination is E3 that was discovered during my graduate studies. As described later by Avram and his gradute student Yuval Reiss, the ligase recognizes several groups of substrates, among them proteins with basic but not with acidic N-termini. Thus, what appeared initially as an artifact turned out to be part of the first specific recognition signal in a target substrate (see below). Parallel to our work on the RNase effect, Avram and Yuval characterized the enzyme and identified on it three distinct substrate binding sites for: (i) basic (the one involved in recognition of basic and Arg-modified acidic Ntermini) and (ii) bulky-hydrophobic N-termini, but also for (iii) larger, yet still undefined “body” sites that reside downstream to the N-terminal residue. Because the enzyme recognized certain substrates at their N()-terminal residue, it was termed E3. In parallel and using a systematic genetic approach in the yeast S. cerevisiae, Alex Varshavsky and his colleagues formulated a general rule (‘N-end rule’) for recognition of all 20 different amino acid residues at the N-terminal site.
Research in the laboratory has evolved also in other directions. We have shown that N--acetylated proteins are also targeted by the ubiquitin system. This important finding demonstrated that this N-terminally modified “family” of proteins, a group that constitutes a large proportion of cellular proteins, must be targeted by signals that are distinct from the N-terminal residue and reside downstream to it: since they do not have free N-termini, they cannot be recognized by this residue. Along with the discovery of the “body” site in E3, we felt that N-terminal recognition involves only a small and limited set of proteins, and the mode of recognition of the numerous substrates of the ubiquitin system must be broad and diverse: they must be recognized by multiple and distinct targeting motifs. At that point, the end of the 1980s, we felt it was time to move from studying model substrates to investigating the fate of specific native cellular substrates. We have shown that an important group of cell regulators – tumor suppressors (e.g. p53) and growth promoters (c-Myc) are targeted by the ubiquitin cell free system. We strongly believed that this must be also true for targeting of these substrates in vivo, which later, through the work of many others and our own, turned out to be the case. We continued and demonstrated that, unlike the paradigm in the field until that time, that degradation of proteins in the lysosome proceeds independently from the ubiquitin system – the two proteolytic pathways are actually linked to one another, and ubiquitination is required for stress-induced lysosomal degradation of cellular proteins. This area has later evolved in a dramatic manner, and engulfed involvement of the ubiquitin system in receptor-mediated endocytosis and autophagy. Other studies involved elucidation of some of the mechanisms involved in the two step ubiquitin-mediated proteolytic activation of the centrally important transcriptional regulator NF-B, demonstration of a role for heat shock proteins in targeting certain protein substrates, and identification of a novel site of ubiquitination – the N-terminal residue of the protein substrate. This modification is clearly different and distinct from recognition of the substrate by E3 at the N-terminal residue. In the latter case, the ligase binds to the N-terminal residue while ubiquitination occurs on an internal lysine residue(s). In N-terminal ubiquitination, modification occurs at the N-terminal residue, while the ligase binds, most probably, to an internal sequence in the protein target molecule. This subject has evolved in a surprising manner and changed another paradigm in the field that ubiquitination is limited to internal lysine(s) of the target substrate; we, and later others, have shown that the phenomenon is not limited to the one protein we identified initially – the muscle-specific transcriptional regulator MyoD, and identified a large group of proteins that undergo N-terminal ubiquitination. This group of proteins contain many that have internal lysine(s), but that from some reason(s) cannot be targeted, but interestingly, also a large group of proteins (such as p16INK4a that plays an important role in cell cycle regulation), that are devoid of any lysine residue. To be degraded by the ubiquitin system, these proteins must undergo N-terminal ubiquitination.
These years have not been simple, however. The Technion has traditionally been a school of engineering, and life sciences and biomedicine have been foreign to many of its senior leaders, faculty members and policy planners: we were treated in many ways like step children, and thoughts of closing the medical school have been aired at times. This deeply rooted philosophy, which only now starts slowly to change, has severely hampered development in these fields and had left the body of researchers and infrastructure in these areas small and battling for survival. Unlike leaders in other schools of engineering like M.I.T. and Caltech, the Technion’s leaders failed to forsee the upcoming revolution in biology and medicine and its huge impact on modern technology. However, through a network of wonderful colleagues all over the world (important among them is my friend Alan Schwartz who is currently at Washington University in St. Louis, but was then at Harvard Medical School; see above for the beginning of our collaboration at the M.I.T.), and fruitful collaborations, I was able to establish an active research group and carry out what I believe was a good and original research program, even under less than optimal, and at times impossible conditions. This was important in balancing my desire to live in Israel, but at the same time to remain at the forefront of the ubiquitin research field that has grown in its importance to become an extremely exciting, yet a highly competitive, area.
Last but not least, I owe a huge debt, which I doubt I shall ever be able to repay, to several people who helped me cross critical stormy waterways along my life. My aunt, Miriam, who took me to her house after the death of my father and made her home a new home for me, enabling me to complete seamlessly my high school studies without any interruption. My brother Yossi (Joseph) and my sister-in-law Atara, who opened their home to me during the fragile times of my high school and medical studies, and made sure I would not collapse along the way, emotionally, but also economically. And last, my wonderful wife Menucha and my son Tzachi (Yitzhak, Isaac; called after my late father). They engulfed me with love, care, and deep understanding of my needs, and were always there for me when I was flying high on the wings of my dreams, not always seeing or listening to them or being with them, physically and emotionally. Without all these wonderful life partners, I could not have achieved anything.
I also owe special thanks to all my mentors, who each contributed in his own way to my upbringing as a scientist. I owe a big debt to Jacob Bar-Tana and the late Benjamin Shapira from the Hebrew University in Jerusalem, who opened for me the gates to the wonderful maze of metabolic pathways, enabling me fall in love with biochemistry. Their enthusiasm and wisdom convinced me, at a critical stage of my development, to pursue a career in biological sciences. Deep thanks to Avram Hershko, with whom I have come a long way in discovering the ubiquitin system, and from whom I learnt the very basic principles of how to approach a scientific problem. I owe special thanks to Ernie Rose for showing me that methodic thinking is not always necessary in science, and is even interfering at times, and that being erratic and disordered, even absent minded, thinking in a most unconventional manner, can yield wonderful ideas and results. The interaction with Ernie is unique, as it gives one a feeling of instability, casting doubt in one’s basic knowledge and beliefs. The real challenge is to select Ernie’s correct idea, which then takes you high above any traditional, step wise approach. Lastly, I owe a huge debt to Harvey Lodish, who is not only a great cell biologist, but a wonderful spiritual mentor in a different way we tend to think of mentors. He gave me complete freedom to choose my own way, but did not let me fall. He always listened carefully and helped me to analyze my results, and with his deep insight was able to find in the ocean of my numbers and graphic data new routes and pathways that I could have never seen or thought of. He used to gently comment on my approach when he felt I got derailed, and helped redirect me. Yet, he was never imposing: Harvey’s active passive educational approach is truly unique. I owe many thanks to all my colleagues, in particular Alan Schwartz, Iasha Sznajder, Yinon Ben-Neriah, and Kazuhiro Iwai, who helped me in many ways along this long voyage. I must also mention my laboratory research associates, initially Sarah Elias (who also helped me in the initial studies) and then Hedva Gonen and Beatrice Bercovich, who have become my eyes and hands since I established my own laboratory. I should mention the major contribution of Hannah Heller, an extremely talented technician of Avram, who was an integral part of our “voyage” and discovery. Dvorah Ganoth and Esther Eythan also helped us along the way, and Clara Segal and Bruvia Rosenberg provided us with skillful technical help. Last but not least, my wonderful graduate students, fellows and visiting scientists, with whom I made new and exciting ways in the rapidly evolving and exciting ubiquitin field.
From Les Prix Nobel. The Nobel Prizes 2004, Editor Tore Frängsmyr, [Nobel Foundation], Stockholm, 2005
This autobiography/biography was written at the time of the award and later published in the book series Les Prix Nobel/Nobel Lectures. The information is sometimes updated with an addendum submitted by the Laureate. To cite this document, always state the source as shown above.