Aaron Ciechanover Research Articles

Dieter Wolf (1941–2023): a life dedicated to understanding protein quality control and the ubiquitin‐proteasome system

Dieter Wolf (1941–2023): a life dedicated to understanding protein quality control and the ubiquitin‐proteasome system

Ubiquitin and its role in proteolisis: the 2004 Nobel prize in chemistry

In the early 1980-s, Aaron Ciechanover, Avram Hershko, and Irwin Rose discovered one of the most important cyclic cellular processes – a regulated ATP-dependent protein degradation, for which they were awarded the 2004 Nobel Prize in Chemistry. These scientists proved the existence of a non-lysosomal proteolysis pathway and completely changed the perception of intracellular protein degradation mechanisms. They demonstrated pre-labelling of a doomed protein in a cell with a biochemical marker called ubiquitin. Polyubiquitylation of a protein as a signal for its proteolysis was a new mechanism discovered as a result of collaborative efforts of three scientists on isolation of enzymes involved in this sequential process, clarification of the biochemical stages, and substantiating the energy dependence mechanism. The article contains biographical data of the Nobel laureates, the methods applied, and the history of the research resulted in the discovery of the phenomenon of proteasomal degradation of ubiquitin-mediated proteins. Keywords: PROTAC, regulated protein degradation, ubiquitin, І. Rose, А. Ciechanover, А. Hershko

Abstract 3541: Epigenetic regulation of KPC1 ubiquitin ligase has a regulatory role on the NF-κB pathway in metastatic melanoma

Abstract Purpose: Abnormal activation of the NF-κB pathway promotes a more aggressive phenotype of cutaneous malignant melanoma. Understanding the mechanisms regulating the NF-κB pathway in melanoma is of critical importance. KPC1 (RNF123) is an E3 ubiquitin ligase that leads to proteasomal processing of precursor NF-κB1 p105 into mature p50, one of the most important steps in the NF-κB pathway regulation. We demonstrated novel epigenetic mechanisms affecting KPC1 expression that lead to an abnormal activation of the NF-κB pathway, which was significant during tumor progression in melanoma patients. Experimental Design: Initially using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including microRNA targeted interaction and DNA methylation. The clinical impact of KPC1 expression and these epigenetic regulations were further assessed in large cohorts of clinically well-annotated melanoma tissues (tissue micro-arrays; n=137, JWCI cohort; n=40) and melanoma TCGA database cohort (n=370). High-throughput RNA sequencing, reverse-phase protein array and human methylation 450k platform were utilized for comprehensive analyses. Results: Initially using metastatic melanoma cell lines, we verified that KPC1 promotes processing of NF-κB1 p105 into p50, thereby modulates NF-κB-target gene expression and suppresses melanoma cell proliferation. Concordantly in melanoma tissue, KPC1 expression was down-regulated in AJCC stage IV melanoma compared to early stages (JWCI cohort stage I/II p=0.013, stage III p=0.004), whereby low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (tissue micro-arrays, n=137, Hazard Ratio 1.810, p=0.006). Furthermore, epigenetic mechanisms regulating KPC1, particularly miR-155-5p and DNA methylation level at its promoter region, was shown to be significant in melanoma lines. This regulatory mechanism was validated in clinical melanoma tissues. High miR-155-5p expression, which is negatively regulated by its promoter DNA methylation level (melanoma TCGA database cohort; Pearson's r -0.455, p<0.001), is significantly associated with KPC1 down-regulation (JWCI cohort; p=0.028, melanoma TCGA database cohort; p=0.003). Conclusions: We identified miR-155-5p, which is epigenetically controlled by its promoter methylation, has a regulatory role on KPC1 expression. These interactions promote to down-regulation of KPC1 and abnormal NF-κB pathway activation, leading to highly proliferative melanoma cells and poor clinical outcomes. These findings suggest utility of KPC1 expression level for stratification of stage IV melanoma patients, and the importance of the miR-155-5p-KPC1-NF-κB-axis in controlling melanoma proliferation. Citation Format: Yuuki Iida, Aaron Ciechanover, Diego M. Marzese, Keisuke Hata, Matias Bustos, Shigeshi Ono, Jinhua Wang, Matthew P. Salomon, Kevin Tran, Yelena Kravtsova-Ivantsiv, Gordon B. Mills, Michael A. Davies, Dave S.B. Hoon. Epigenetic regulation of KPC1 ubiquitin ligase has a regulatory role on the NF-κB pathway in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3541.

Chemistry in Israel-at a Crossroads?

Such achievements are the fruits of the inspired and incisive leadership that served as the foundation of the academic world in my country—the manifestations of an early vision that has led to what we have come to know as modern Israel. David Ben-Gurion, undoubtedly one indefatigable agent of progress, as well as the country s inaugural Prime Minister, wrote two years after the creation of the State: “We are a small and poor nation, and our country has suffered neglect and devastation. This imposes a severe austerity upon us, a condition, which may last a long time. Strangely enough, these very facts forbid us austerity in two areas, in science and technology...Only if we were able to use the attainments of scientific and technological developments—can we succeed”.

A conversation with Aaron Ciechanover

For decades, the attention of the scientific community was focused on the central dogma of biology — the decoding of the genetic information embedded in DNA. Little research was dedicated to how proteins are degraded and removed from cells. Enter onto the scene a young graduate student, Aaron Ciechanover, who with his mentor Avram Hershko, uncovered the complex and elegant ubiquitin proteolytic system. For his discovery, Ciechanover (Figure ​(Figure1)1) shared in the 2004 Nobel Prize in Chemistry with Hershko and Irwin Rose. The complete interview, with more stories about being a member of the Pontifical Academy of Sciences, the magic of “A-ha” moments, and the conflict between religion and Darwinism, can be seen on the JCI website, http://www.jci.org/kiosk/cgm.

Ubiquitin: Molecular modeling and simulations

The synthesis and destruction of proteins are imperative for maintaining their cellular homeostasis. In the 1970s, Aaron Ciechanover, Avram Hershko, and Irwin Rose discovered that certain proteins are tagged by ubiquitin before degradation, a discovery that awarded them the 2004 Nobel Prize in Chemistry. Compelling data gathered during the last several decades show that ubiquitin plays a vital role not only in protein degradation but also in many cellular functions including DNA repair processes, cell cycle regulation, cell growth, immune system functionality, hormone-mediated signaling in plants, vesicular trafficking pathways, regulation of histone modification and viral budding. Due to the involvement of ubiquitin in such a large number of diverse cellular processes, flaws and impairments in the ubiquitin system were found to be linked to cancer, neurodegenerative diseases, genetic disorders, and immunological disorders. Hence, deciphering the dynamics and complexity of the ubiquitin system is of significant importance. In addition to experimental techniques, computational methodologies have been gaining increasing influence in protein research and are used to uncover the structure, stability, folding, mechanism of action and interactions of proteins. Notably, molecular modeling and molecular dynamics simulations have become powerful tools that bridge the gap between structure and function while providing dynamic insights and illustrating essential mechanistic characteristics. In this study, we present an overview of molecular modeling and simulations of ubiquitin and the ubiquitin system, evaluate the status of the field, and offer our perspective on future progress in this area of research.

Ubiquitin and Drug Discovery: Challenges and Opportunities

In 1977 ATP-dependent protein degradation in reticulocytes was demonstrated by Alfred Goldberg. Beginning in 1978, the ATP-dependent ubiquitin system for proteolysis was described by Avram Hershko, Irwin Rose and Aaron Ciechanover and colleagues, with the proteasome being characterized soon thereafter. Before this, the prevailing concept was that whatever protein degradation was taking place in cells was through lysosomes.

Translating experience: Thinking outside the box

Tony Hunter is Director of the Salk Institute Cancer Center, 10010 N Torrey Pines Road, San Diego, California 92037, USA. e-mail: hunter@salk.edu acetyl group. Had we been primed to think outside the box, we might have discovered this initiation mechanism sooner. In 1970, Richard and I were using rabbit reticulocytes to analyse the fate of nascent globin chains released prematurely from ribosomes following puromycin treatment. We found that these chains were rapidly degraded; this also occurred in vitro when isolated ribosomes incubated with puromycin were added to a reticulocyte lysate supplemented with ATP. Just before I left for my postdoctoral position at the Salk Institute, we presented these results at a 1971 meeting and, in a proceedings chapter, speculated that proteases unable to recognize intact folded globin chains degraded these truncated proteins. Had we continued the project and thought of alternative explanations, we might have uncovered the ATP-dependent protease activity, reported by Fred Goldberg in 1977 and later shown to be due to the proteasome, and the ubiquitin-mediated protein degradation system, reported by Avram Hershko and Aaron Ciechanover in 1978 — both using rabbit reticulocyte lysates. In 1973, when I returned to the Department of Biochemistry in Cambridge following my postdoc, a new willingness to think laterally led to an unexpected discovery. When a fire destroyed our lab in June 1974, we became refugees and were graciously offered temporary space in the New Addenbrookes site, opposite the MRC Laboratory of Molecular Biology (LMB). Our plight led Max Perutz, the Director of LMB, to offer us dining rights in the LMB cafeteria, where we ate lunch every day. The unspoken rule of the cafeteria was that you had to sit with people from other groups, and this soon led to Tim Hunt and myself collaborating with John Knowland and David Zimmern in the LMB tobacco mosaic virus (TMV) group, led by Aaron Klug. At the time, the packaged TMV genomic RNA was known to encode the 18 kDa viral coat protein, but all attempts to translate coat protein in vitro had failed, generating instead much larger 140–160 kDa products. We found the same when we translated TMV virion RNA in the wheat germ system we were using to study protein synthesis initiation, or in Xenopus laevis oocytes. Eventually, we realized that as TMV-infected tobacco plants make large amounts of coat protein, their leaves must contain a coat protein mRNA. One of the first experiments we did in our new home was to translate total RNA isolated from TMV-infected tobacco leaves using wheat germ samples stored in a liquid N2 freezer that had survived the fire. Gratifyingly, we obtained authentic coat protein, and demonstrated that its mRNA corresponded to the 750 bases at the 3' end of the genomic RNA. We had discovered that TMV expresses its coat protein through a subgenomic viral RNA, a strategy commonly adopted by RNA viruses for expressing individual proteins from polycistronic RNAs. Our findings did not explain why fragments from the 3' end of the genomic RNA failed to translate coat protein. This mystery was solved in 1975, when a 5' methylated cap structure was shown to be required for eukaryotic mRNA translation. This initiation mechanism restricts translation to the cistron adjacent to the cap, explaining why internal cistrons, such as the coat protein gene, cannot be translated. We now know that the virion RNA has a cap, as does the subgenomic RNA made in infected cells. These early experiences stood me in good stead when I fortuitously stumbled across a novel phospho-amino acid generated in an in vitro kinase assay using polyoma virus middle T antigen. This was not the expected phospho-serine or phospho-threonine, and only by being willing to ignore dogma did I surmise that this might be phospho-tyrosine. The rest, as they say, is history.

An interview with Aaron Ciechanover

An interview with Aaron Ciechanover

The 2008 Lindau Nobel Laureate Meeting: Aaron Ciechanover, Chemistry 2004

Aaron Ciechanover was born in Haifa, Israel in October 1947. He shared the Nobel Prize in Chemistry in 2004 with Avram Hershko and Irwin Rose for their discovery of ubiquitin-mediated protein degradation. When Ciechanover began his work on proteolysis, the field was outside the realm of scientific mainstream as many thought that the fundamental secrets relating to sequence specificity were relevant to the synthetic side, or code side. The notion that specific sequences could selectively guide a destructive process did not naturally occur to scientists including Ciechanover himself. The emergence of controversial evidence demonstrating a requirement for metabollic energy in intracellular protein degradation, refuted the idea that cellular proteolysis was an entirely exergonic process occuring in the lysosme and prompted Ciechanover, Hershko, and Rose to "launch an attack" on the system, in order to uncover true pathway. Later findings of Ciechanover and subsequent groups showed that not only was the process energy-dependent, but that 8% of the human genome is remarkabley one large ubiquitin system. Following the recapitulation and reflection of his work, Ciechanover shares insights into his principal and philosophical approach to science and life alltogether. The life and work of Aaron Ciechanover are deeply rooted and influenced by Judaism and Israel and it is therefore that with only brief intermission, Ciechanover spent his scientific career in Israel as he is--through his presence and work--able to contribute and shape presence and future of the State of Israel.

Interview with Aaron Ciechanover

Interview with Aaron Ciechanover

POEM 2008 makes its debut a success

On November 24–27th 2008, the first Photonics and Optoelectronics Meetings (POEM 2008), organized by Wuhan National Laboratory for Optoelectronics (WNLO), was held in Wuhan Science and Technology Convention and Exhibition Center, Wuhan, China. The POEM 2008, in conjunction with the 8th International Symposium on Optical Storage/2008 International Workshop on Information Data Storage (ISOS/IWIDS 2008), and the International Conference on Photonics and Imaging in Biology and Medicine (PIBM 2008), consists of a plenary session, six parallel sessions, industry seminars, technology and talent exchange, and a consulting seminar of developing strategy. It has four conference chairs: Dr. Chaohui Ye, Director of WNLO and member of the Chinese Academy of Sciences (CAS); Dr. Bingkun Zhou, Professor of Tsinghua University, member of CAS; Dr. Britton Chance, Academician of six countries, Professor of the University of Pennsylvania and 2008 Friendship Award Winner of the Chinese Government; and Dr. Victor V. Apollonov, member of the Russian Academy of Sciences and Director of A. M. Prohorov General Physics Institute. There are 12 academicians from home and abroad attending the conference including Dr. Aaron Ciechanover, 2004 winner of the Nobel Prize in chemistry, Dr. Bernd Wilhelmi, member of Germany Leopodina and the former Soviet Union Academy of Sciences, Dr. Paul Ching-wu Chu, president of Hong Kong University of Science and Technology, the U.S. chief scientist in physics of the University of Houston, academician of three countries, etc. On November 24th, about 10 national and local governmental leaders attended the plenary session and 5 worldrenown experts, including Dr. Aaron Ciechanover, Dr. Victor V. Apollonov, Dr. Dieter Stefan Jager, Prof. Ian H. White, and Prof. Xicheng Zhang gave presentations. Also, Dr. Paul Ching-wu Chu was invited to give a talk on “The spirit of science and practice” and Prof. TsutomuMiyasaka, Director of the Graduate School of Engineering of Toin University of Yokohama gave a report in WNLO. Over 600 representatives from more than 10 countries attended the conference including 30 people from abroad. To promote the construction and development of the WNLO, strengthen the connection between the WNLO and “Wuhan $Optics Valley of China (WOVC)”, the “Consulting Seminar of Developing Strategy on WOVC and WNLO” was held in the afternoon of November 24th. Well-known scholars, business executives and department leaders from home and abroad discussed the overall plan and present status of the industry development of WOVC, the interactive relationship between WOVC and WNLO and layout of the developing strategy of WNLO. During the meeting, a cooperation agreement on the Wuhan East Lake High-Tech Development Zone supporting the development of WNLO was signed. The conference mainly focused on six topics — fiber optical communication and sensors, laser technology and applications, optical storage and new storage technology, optoelectronic devices and integration, Tera-Hertz science and technology, and photonics and imaging in biology and medicine. Rich content of oral and invited presentations, plenary discussion, short courses, hot topics, best student award and student-focused poster session were presented on the conference. Latest achievements on the frontier of the field are published on the conference. On November 25th, experts of optical data storage attended a new product’s release meeting — Next-generation Versatile Disk (NVD). A new generation red light high-definition products from WOVC will be on the market for sale soon. 734 papers including 348 oral presentations and 386 posters were contributed. At noon of November 27th, after a

A Proteasomal ATPase Contributes to Dislocation of Endoplasmic Reticulum-associated Degradation (ERAD) Substrates

Endoplasmic reticulum (ER)-associated degradation (ERAD) eliminates aberrant proteins from the ER by dislocating them to the cytoplasm where they are tagged by ubiquitin and degraded by the proteasome. Six distinct AAA-ATPases (Rpt1-6) at the base of the 19S regulatory particle of the 26S proteasome recognize, unfold, and translocate substrates into the 20S catalytic chamber. Here we show unique contributions of individual Rpts to ERAD by employing equivalent conservative substitutions of the invariant lysine in the ATP-binding motif of each Rpt subunit. ERAD of two substrates, luminal CPY*-HA and membrane 6myc-Hmg2, is inhibited only in rpt4R and rpt2RF mutants. Conversely, in vivo degradation of a cytosolic substrate, DeltassCPY*-GFP, as well as in vitro cleavage of Suc-LLVY-AMC are hardly affected in rpt4R mutant yet are inhibited in rpt2RF mutant. Together, we find that equivalent mutations in RPT4 and RPT2 result in different phenotypes. The Rpt4 mutation is manifested in ERAD defects, whereas the Rpt2 mutation is manifested downstream, in global proteasomal activity. Accordingly, rpt4R strain is particularly sensitive to ER stress and exhibits an activated unfolded protein response, whereas rpt2RF strain is sensitive to general stress. Further characterization of Rpt4 involvement in ERAD reveals that it participates in CPY*-HA dislocation, a function previously attributed to p97/Cdc48, another AAA-ATPase essential for ERAD of CPY*-HA but dispensable for proteasomal degradation of DeltassCPY*-GFP. Pointing to Cdc48 and Rpt4 overlapping functions, excess Cdc48 partially restores impaired ERAD in rpt4R, but not in rpt2RF. We discuss models for Cdc48 and Rpt4 cooperation in ERAD.

2006 SCBA KEYNOTE SPEAKERS

2006 SCBA Keynote Speakers are as follow: Yuh-Nung Jan, Ph. D; Lily Jan, Ph. D.; Aaron Ciechanover, M.D., D.Sc.; Peter Courtland Agre, M.D.; Yi Zhang, Ph.D.; Yuet Wai Kan, M.B.B.S. (Hons), D.Sc.; Shu Chien, M.D., Ph.D.; Allen Chao, Ph.D. and Nancy T. Chang.

The discovery of ubiquitin-dependent proteolysis

In early 1980, Irwin A. Rose, Avram Hershko, and Aaron Ciechanover published two papers in PNAS that reported the astounding observation that energy-dependent intracellular proteolysis was far more complicated than the previously accepted models of lysosomal proteolysis or the action of ATP-dependent proteases such as bacterial lon. In fact, it has turned out to be even more complicated than they could have suspected. The general model of covalently attaching a small protein as a targeting signal has proved to be every bit as important to eukaryotic cells as the better understood modifications such as phosphorylation or acetylation. The key player in this modification, a small protein called ubiquitin (APF-1 in these papers), is the founding member of a large family of proteins containing the beta-grasp fold and is used as a posttranslational targeting signal to modify the structure, function, and/or localization of other proteins. The story of this discovery is a textbook example of the confluence of intellectual curiosity, unselfish collaboration, chance, luck, and preparation.

The Ubiquitin System for Protein Degradation and Some of Its Roles in the Control of the Cell‐Division Cycle (Nobel Lecture)

Protein breakdown is an exact, controlled process in which the protein molecule ubiquitin plays a decisive role by binding to a protein and labeling it for breakdown. The cellular mechanisms of this process are widely known today thanks to the work of the 2004 Nobel Laureates Irwin Rose, Avram Hershko, and Aaron Ciechanover. The Laureates report at first hand in their Nobel Lectures.chemical structure image

Ubiquitin at Fox Chase (Nobel Lecture)

Protein breakdown is an exact, controlled process in which the protein molecule ubiquitin plays a decisive role by binding to a protein and labeling it for breakdown. The cellular mechanisms of this process are widely known today thanks to the work of the 2004 Nobel Laureates Irwin Rose, Avram Hershko, and Aaron Ciechanover. The Laureates report at first hand in their Nobel Lectures.

Discovery of the ubiquitin proteasome system and its involvement in apoptosis

This special issue of Cell Death and Differentiation has come to fruition thanks to the generous assistance of the Nobel Foundation in Stockholm, http://www.nobelprize.org (©The Nobel Foundation 2004). It is an honour and privilege to write a short introductory editorial to lectures1, 2, 3 and interviews4, 5, 6 with Aaron Ciechanover, Avram Hershko and Irwin Rose, the 2004 Nobel Laureates in Chemistry, for their pioneering work on the discovery of ubiquitin-mediated protein degradation. This is an additional tribute to these scientists, in recognition of their discovery and characterisation of the ubiquitin proteasome system (UPS), made in a small laboratory by great scientists with limited material, but not intellectual, resources. A detailed disquisition of the discovery of the UPS may be found in the very recent review published by Ciechanover.7 A brief, limited timeline covering the identification of the UPS is shown in Figure 1.

Early work on the ubiquitin proteasome system, an interview with Aaron Ciechanover

Early work on the ubiquitin proteasome system, an interview with Aaron Ciechanover

Nobel Committee Tags Ubiquitin for Distinction

It must be very difficult to serve on the committee to select Nobel Prize winners—to have to select one or two scientific advances amidst many outstanding discoveries, to have to choose the two or three key investigators (who may not be the most visible or most cited ones), and to have to sift through many nominations that extol the achievements of some candidates (but overlook those of others). Nevertheless, the scientists selected in recent years have consistently been ones who have made outstanding contributions, and this year’s decision to award the Nobel Prize for Chemistry to Avram Hershko, Aaron Ciechanover, and Irwin Rose for the discovery of the ubiquitin system is particularly insightful and honors three biochemists whose seminal work has altered modern biology.