Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Classic Articles:
Ciechanover et al. 255 (16): 7525
Hershko et al. 257 (23): 13964
Hershko et al. 258 (13): 8206

J. Biol. Chem., Vol. 281, Issue 40, 32, October 6, 2006
This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kresge, N.
Right arrow Articles by Hill, R. L.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Kresge, N.
Right arrow Articles by Hill, R. L.
Related Collections
Right arrow Classic Articles
Ciechanover et al. 255 (16): 7525
Hershko et al. 257 (23): 13964
Hershko et al. 258 (13): 8206
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Classics

The Discovery of Ubiquitin-mediated Proteolysis by Aaron Ciechanover, Avram Hershko, and Irwin Rose

Nicole Kresge, Robert D. Simoni, and Robert L. Hill

Characterization of the Heat-stable Polypeptide of the ATP-dependent Proteolytic System from Reticulocytes
(Ciechanover, A., Elias, S., Heller, H., Ferber, S., and Hershko, A. (1980) J. Biol. Chem. 255, 7525–7528)

Immunochemical Analysis of the Turnover of Ubiquitin-Protein Conjugates in Intact Cells. Relationship to the Breakdown of Abnormal Proteins
(Hershko, A., Eytan, E., Ciechanover, A., and Haas, A. L. (1982) J. Biol. Chem. 257, 13964–13970)

Components of Ubiquitin-Protein Ligase System. Resolution, Affinity Purification, and Role in Protein Breakdown
(Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206–8214)

In a series of pioneering biochemical studies in the late 1970s and early 1980s, Avram Hershko, Aaron Ciechanover, and Irwin A. Rose discovered and characterized the ATP-dependent, ubiquitin-mediated protein degradation system. Part of this work was done during a series of sabbatical leaves when Hershko and Ciechanover worked in Rose's laboratory at the Fox Chase Cancer Center in Philadelphia.

Hershko received his M.D. (1965) and his Ph.D. (1969) from the "Hadassah" Faculty of Medicine of the Hebrew University in Jerusalem and then did a postdoctoral fellowship with Gordon Tomkins at the University of California in San Francisco. There, he studied the degradation of tyrosine aminotransferase. Hershko's results confirmed and extended earlier findings by Melvin Simpson on the energy dependence of the liberation of amino acids from proteins.

In 1971 Hershko returned to Israel and became Chairman of Biochemistry in the Faculty of Medicine of the Technion, the Israel Institute of Technology. He continued to pursue the problem of how proteins are degraded in cells, and why energy is required for this process. For a number of years, he tried to establish a cell-free system that reproduced energy-dependent protein degradation. Finally, he settled on a reticulocyte cell-free crude extract established by Etlinger and Goldberg (1). At that time, Ciechanover joined Hershko's laboratory as a graduate (D.Sc.) student, after earning a Master of Science in 1971 and an M.D. in 1974 from the "Hadassah" Medical School of the Hebrew University in Jerusalem.

In their initial attempt to characterize the ATP-dependent activity in the reticulocyte crude extract, Ciechanover and Hershko showed, already early on, that unlike "classical" proteases that digest their substrates directly, the reticulocyte "protease" contained at least two complementing fractions, I (the active component of which was shown to be a small heat-stable protein designated initially as APF-1-ATP-dependent proteolysis factor 1, later identified as ubiquitin) and II, which were required together to reconstitute the activity observed in the crude extract (2). This was an important observation not only because the initial characterization of ubiquitin started at that point but also because from that stage on classical biochemistry of reconstitution was employed each time the proteolytic activity was lost during a purification step, leading to the discovery of the two-step novel mode of action of the system—initial substrate tagging of the target substrate by ubiquitin, followed by its subsequent degradation with recycling of ubiquitin.GoGoGo


Figure 1
View larger version (177K):
[in this window]
[in a new window]
  		Avram Hershko. Photo courtesy of Technion-Israel Institute of Technology. Photographed by Miki Koren.

 


Figure 2
View larger version (162K):
[in this window]
[in a new window]
  		Aaron Ciechanover. Photo courtesy of Technion-Israel Institute of Technology. Photographed by Miki Koren.

 


Figure 3
View larger version (126K):
[in this window]
[in a new window]
  		Irwin Rose. Photo courtesy of the University of California Irvine.

 
In 1976, Hershko attended a Fogarty Conference on Regulation at the National Institutes of Health. Rose also attended this meeting, and the two met for the first time and learned of their mutual interest in protein breakdown. Rose's interest in the field began in 1955 when he became an Instructor in Biochemistry at Yale University Medical School and met Simpson. He had just completed postdoctoral fellowships at Case Western Reserve University with C. E. Carter and at New York University with Journal of Biological Chemistry (JBC) Classic author Severo Ochoa (3) after earning his B.S. (1948) and his Ph.D. in biochemistry (1952) from the University of Chicago.

Hershko had a sabbatical year in 1977 and decided to spend it with Rose who had been at The Institute for Cancer Research at the Fox Chase Cancer Center since 1963. Hershko brought Ciechanover with him, and the three scientists started working on elucidating the protein degradation system.

Hershko, Ciechanover, and Rose reported that the second fraction of the reticulocyte lysate could be further subdivided into an ATP-stabilized protein of approximately 450 kDa (most probably containing the 26 S proteasome) and a fraction that contained the E1–E3 conjugating enzymes that were later isolated (4). All three fractions were required to degrade 125I-labeled albumin. Soon, they discovered that multiple APF-1 molecules are bound covalently to target substrates in the lysate and mark them for degradation by a protease that recognizes only conjugated proteins. APF-1 is recycled during the process (5, 6).

Ciechanover and Hershko subsequently purified and characterized APF-1 from rabbit reticulocytes. This is the subject of the first JBC Classic reprinted here. An amino acid analysis of APF-1, along with its known molecular mass and other general characteristics raised the strong suspicion that APF-1 was ubiquitin.Go


Figure 4
View larger version (8K):
[in this window]
[in a new window]
  		Proposed sequence of events in the ubiquitin-protein ligase system.

 
Ubiquitin was originally isolated by Gideon Goldstein and co-workers in a search for hormones of the thymus and was subsequently found to be present in all tissues and organisms, hence its name (7). Yet, later studies have demonstrated that ubiquitin is present only in eukaryotes and it lacks any hormonal activity, so its functions had remained elusive at the time (8). An important discovery however was that ubiquitin is conjugated in an isopeptide bond to an internal lysine residue in histone H2A (9, 10).

The connection between APF-1 and ubiquitin was made by three postdoctoral fellows at Fox Chase who worked there in parallel to Hershko and Ciechanover and followed the development of the system. Keith Wilkinson and Arthur Haas were from the Rose laboratory, and Mike Urban was from Martin Nemer's laboratory. Urban was working with chromatin and noted that similar to APF-1, ubiquitin was also a small protein that was linked covalently to other proteins, in this case histones. As described in the first JBC Classic, the size and amino acid composition of APF-l and ubiquitin were in agreement, suggesting that the two proteins were the same. This final identification was published in a paper (11) that appeared back to back with the Ciechanover and Hershko JBC Classic.

This ubiquitin tagging hypothesis received support when Hershko and Ciechanover observed a marked increase in the labeling of ubiquitin-protein conjugates during the formation of abnormal, rapidly degrading proteins in reticulocytes and nucleated eukaryotic cells. This is the subject of the second JBC Classic reprinted here. In this paper Hershko and Ciechanover developed an immunochemical method for isolating ubiquitin-protein conjugates from intact cells. Cells were incubated in the presence of amino acid analogues to generate unstable, rapidly degrading proteins and pulse-labeled with tryptophan, an amino acid that is missing in ubiquitin. Using an antibody against ubiquitin, they showed a direct correlation between the instability of the synthesized proteins and the level of the corresponding ubiquitin conjugates generated: degradation of the short lived proteins was accompanied by a corollary generation of a high level of ubiquitin adducts. Thus, it was clear that ubiquitination was not restricted to terminally differentiating reticulocytes but was also observed in the Ehrlich ascites cells used, indicating the universal nature of the system.

Between 1981 and 1983, Hershko and Ciechanover worked out the multistep ubiquitin-tagging hypothesis by isolating and characterizing the enzymes E1, E2, and E3. They first purified the ubiquitin-activating enzyme E1 by affinity chromatography using Sepharose-immobilized ubiquitin (12). E1 bound covalently to ubiquitin and was eluted by AMP and PPi, reversing the reaction of its activation and binding to ubiquitin. d1 Realizing that E1 could not form ubiquitin-protein conjugates by itself, Ciechanover and Hershko used their affinity chromatography strategy to purify the ubiquitin carrier protein E2 (by using dithiothreitol, reversing the mechanism of E2 binding to the immobilized ubiquitin via thiol ester formation) and the ubiquitin-protein ligase E3 (by using high salt or high pH). This purification and subsequent analysis of the roles of the three enzymes in the three-step cascade mechanism of ubiquitin activation and conjugation is reported in the third JBC Classic reprinted here.

Hershko and Ciechanover found that the binding of E2 to the immobilized ubiquitin required E1 and ATP, but the binding of E3 to the column required neither. Taken together, the results indicated that E2 bound covalently to the column like E1 did, whereas E3, which could be eluted by salt/high pH, bound non-covalently. Based on their observations, they hypothesized that E1 transferred ubiquitin to E2, which then transferred ubiquitin to the substrate in the presence of E3 (Fig. 4). Although they used a single E3 and a single model substrate, they predicted that the specificity of the E3 component determined which proteins in the cell were marked for destruction, determining the high specificity of the system toward its potential numerous substrates. Thus the "the multistep ubiquitin-tagging hypothesis" was born.

As a result of their pioneering studies on ubiquitin-mediated proteolysis, Hershko, Ciechanover, and Rose were awarded the 2004 Nobel Prize in Chemistry "for the discovery of ubiquitin-mediated protein degradation."

Hershko remained at the Israel Institute of Technology where he is currently a Distinguished Professor at the Rappaport Family Institute for Research in Medical Sciences as well as an Adjunct Professor of Pathology at New York University. In addition to the Nobel Prize, he has received many honors including the 1987 Weizmann Prize for Sciences, the 1994 Israel Prize in Biochemistry and Medicine, the 1999 Gairdner International Award, the 2000 Alfred P. Sloan Prize, the 2000 Albert Lasker Award for Basic Medical Research, the 2001 Merck Award from the American Society for Biochemistry and Molecular Biology, the 2001 Wolf Prize for Medicine, the 2002 E. B. Wilson Medal from the American Society of Cell Biology, and the 2005 Stein and Moore Award from the Protein Society.1

Ciechanover completed his doctorate in science (D.Sc.) in the Technion, after which he did a postdoctoral fellowship with Harvey F. Lodish in the Department of Biology and the Whitehead Institute at M.I.T. He then joined the Faculty of Medicine at the Technion, where he is currently a Distinguished Professor in the Cancer and Vascular Biology Research Center of the Rappaport Institute, and a Visiting Professor in the Department of Medicine in the Feinberg Medical School at Northwestern University in Chicago and the Department of Pediatrics at Washington University School of Medicine in St. Louis. In addition to the Nobel Prize, Ciechanover received the Austria Innsbruck University Wachter Prize in 1999, the Albert Lasker Award for Basic Medical Research in 2000, the Israel Prize for Biology in 2003, and the Japanese Society for Promotion of Science (JSPS) Distinguished Scientist Award in 2003.2

Rose remained at Fox Chase until his retirement in 1997. Following this, he moved to Laguna Woods in Southern California where he was able to continue his laboratory work using space and facilities at the University of California, Irvine. He is currently a Distinguished Professor-in-Residence at the Department of Physiology and Biophysics of the College of Medicine. Rose was elected to the National Academy of Sciences in 1979 and received the Philadelphia Section Award from the American Chemical Society in 1975, and the Washington State University's Regents' Distinguished Alumnus Award in 2005 (Rose attended Washington State University for 1 year prior to serving in the Navy during WWII).3

FOOTNOTES

1 Biographical information on Avram Hershko was taken from Refs. 13 and 14. Back

2 Biographical information on Aaron Ciechanover was taken from Refs. 15 and 16. Back

3 Biographical information on Irwin Rose was taken from Refs. 17 and 18. Back


REFERENCES

  1. Etlinger, J. D., and Goldberg, A. L. (1977) A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc. Natl. Acad. Sci. U. S. A. 74, 54–58[Abstract/Free Full Text]
  2. Ciechanover, A., Hod, Y., and Hershko, A. (1978). A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105[CrossRef][Medline] [Order article via Infotrieve]
  3. JBC Classics: Stern, J. R., and Ochoa, S. (1951) J. Biol. Chem. 191, 161–172; Korkes, S., del Campillo, A., Gunsalus, I. C., and Ochoa, S. (1951) J. Biol. Chem. 193, 721–735 (http://www.jbc.org/cgi/content/full/280/11/e8); Salas, M., Smith, M. A., Stanley, W. M., Jr., Wahba, A. J., and Ochoa, S. (1965) J. Biol. Chem. 240, 3988–3995 (http://www.jbc.org/cgi/content/full/281/21/e16)
  4. Hershko, A., Ciechanover, A., and Rose, I. A. (1979) Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP. Proc. Natl. Acad. Sci. U. S. A. 76, 3107–3110[Abstract/Free Full Text]
  5. Ciechanover, A., Heller, H., Elias, S., Haas, A. L., and Hershko, A. (1980) ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation. Proc. Natl. Acad. Sci. U. S. A. 77, 1365–1368[Abstract/Free Full Text]
  6. Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980) Proposed role of ATP in protein breakdown: conjugation of proteins with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc. Natl. Acad. Sci. U. S. A. 77, 1783–1786[Abstract/Free Full Text]
  7. Goldstein, G., Scheid, M., Hammerling, U., Boyse, E. A., Schlesinger, D. H., and Niall, H. D. (1975) Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells. Proc. Natl. Acad. Sci. U. S. A. 72, 11–15[Abstract/Free Full Text]
  8. Low, T. L. K., and Goldstein, A. L. (1979) The chemistry and biology of thymosin: amino acid analysis of thymosin {alpha}1 and polypeptide beta1. J. Biol. Chem. 254, 987–995[Abstract/Free Full Text]
  9. Goldknopf, I. L., and Busch, H. (1977) Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24. Proc. Natl. Acad. Sci. U. S. A. 74, 864–868[Abstract/Free Full Text]
  10. Hunt, L. T., and Dayhoff, M. O. (1977) Amino-terminal sequence identity of ubiquitin and the non-histone component of nuclear protein A24. Biochem. Biophys. Res. Commun. 74, 650–655[CrossRef][Medline] [Order article via Infotrieve]
  11. Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980) Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes. J. Biol. Chem. 255, 7529–7532[Abstract/Free Full Text]
  12. Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) "Covalent affinity" purification of ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542[Abstract/Free Full Text]
  13. Hershko, A. (2005) Avram Hershko—autobiography. In The Nobel Prizes 2004 (Frängsmyr, T., ed) Nobel Foundation, Stockholm
  14. Hershko, A. (2005) The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. In The Nobel Prizes 2004 (Frängsmyr, T., ed) Nobel Foundation, Stockholm
  15. Ciechanover, A. (2005) Aaron Ciechanover—autobiography. In The Nobel Prizes 2004 (Frängsmyr, T., ed) Nobel Foundation, Stockholm
  16. Ciechanover, A. (2005) Intracellular protein degradation: from a vague idea through the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. In The Nobel Prizes 2004 (Frängsmyr, T., ed) Nobel Foundation, Stockholm
  17. Rose, I. A. (2005) Irwin Rose—autobiography. In The Nobel Prizes 2004 (Frängsmyr, T., ed) Nobel Foundation, Stockholm
  18. Rose, I. A. (2005) Ubiquitin at Fox Chase. In The Nobel Prizes 2004 (Frängsmyr, T., ed) Nobel Foundation, Stockholm

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kresge, N.
Right arrow Articles by Hill, R. L.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Kresge, N.
Right arrow Articles by Hill, R. L.
Related Collections
Right arrow Classic Articles
Ciechanover et al. 255 (16): 7525
Hershko et al. 257 (23): 13964
Hershko et al. 258 (13): 8206
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement