Protacs: Chimeric Molecules That Target Proteins to the Skp1–Cullin–F Box Complex for Ubiquitination and Degradation
Proceedings of the National Academy of Sciences, 2001 · paper · Chemistry
Describes Protac-1, a two-part molecule linking an IκBα phosphopeptide to ovalicin to recruit the enzyme MetAP-2 to the SCF-β-TRCP ubiquitin ligase. MetAP-2 was ubiquitinated and degraded in a Protac-dependent manner, suggesting a way to target disease proteins.
Checked: PDF from europepmc.org, fetched 4 Oct 2026. Page numbers are those of that PDF.
11 findings
Substance Methods give OVA in the Xenopus extract reactions as "in addition to OVA (10 µM final)", whereas the Fig. 5 legend says the extract was "fortified with OVA (100 µM)".
- p. 2 Methods, Degradation Experiments with Xenopus Extracts
was added to 10
- p. 2 Methods, Degradation Experiments with Xenopus Extracts
in addition to OVA (10
- p. 4 Fig. 5 legend
fied with OVA (100
- p. 2 Methods, Degradation Experiments with Xenopus Extracts
OVA (10 mM final)
Only the authors can say what is right here.
Judge's note: Read on the page 2 and page 4 images; the unit is µM in both places (the text layer shows mM because µ is lost). The other additions agree between Methods and legend (IKK-EE 0.4 µg, OA 10 µM, LLnL 50 µM, epoxomicin 10 µM), so the same reaction is described. The Methods value is labelled "final"; the legend gives no stock/final qualifier and no statement elsewhere reconciles a tenfold difference. Only the authors can say which is right.
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Substance For the Fig. 2A binding experiment the Results say "we incubated MetAP-2 (18 µM) with increasing concentrations of Protac-1 (Fig. 2A)", whereas the Fig. 2 legend says "MetAP-2 (9 µM) was incubated with increasing concentrations of Protac-1" and the Methods say "MetAP-2 (9 µM) was incubated with increasing concentrations of Protac-1".
- p. 3 Results, 'MetAP-2 Specifically Binds Protac-1 in Vitro'
we incubated MetAP-2 (18
- p. 3 Fig. 2 legend (A)
MetAP-2 (9
- p. 2 Methods, MetAP-2–Protac-1 Coupling Assay
MetAP-2 (9
- p. 3 Results, MetAP-2 Specifically Binds Protac-1
we incubated MetAP-2 (18 mM) with increasing con-
Only the authors can say what is right here.
Judge's note: All three read on the page 2 and page 3 images; units are µM. The same incubation is described in the same sentence form with 18 µM in one place and 9 µM in two. A possible explanation is that 18 µM is the stock and 9 µM the concentration after 1:1 mixing (the ubiquitination Methods use 4 µl of 18 µM MetAP-2 plus 4 µl of Protac-1, and the Fig. 3 Methods call the result a 9 µM/50 µM mixture), but the paper does not say this for the coupling assay and the Results sentence gives 18 µM as the incubation concentration. Authors should say which is meant.
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Substance For Fig. 4C lane 7 the Results say "unlinked OVA (50 µM) plus IPP (50 µM) were not able to substitute for the OVA–IPP conjugate (lane 7)", whereas the Fig. 4 legend says "In lane 7, unlinked IPP and OVA were added at 100 µM in place of Protac-1".
- p. 3 Results, 'Protac-1 Mediates the Ubiquitination of MetAP-2', last lines of page
unlinked OVA (50
- p. 4 Fig. 4 legend (C)
unlinked IPP and OVA were added at 100
- p. 3 Results, Protac-1 Mediates the Ubiquitination
unlinked OVA (50 mM) plus IPP (50 mM) were not
- p. 4 Fig. 4 legend, C
unlinked IPP and OVA were added at 100 mM in place of Protac-1
Only the authors can say what is right here.
Judge's note: Read on the page 3 and page 4 images; units are µM in both. Same lane, two concentrations. The Methods give competitor concentrations as "100 µM final" for the competition experiments (Fig. 4D), which matches the legend's figure, and no stock/final distinction is stated for lane 7. Reading the legend's 100 µM as a total of 50 + 50 is possible but not what it says. Only the authors can say which is right.
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Reference or label The sentence "these immunoprecipitates promoted ubiquitination of IKK-phosphorylated glutathione S-transferase-IκBα in a manner that was inhibited by the IPP and Protac-1 (data not shown; ref. 7)" cites reference 7 ("Sin, N., Meng, L., Wang, M. Q., Wen, J. J., Bornmann, W. G. & Crews, C. M. (1997) Proc. Natl. Acad. Sci. USA 94, 6099–6103"), which the Introduction cites for MetAP-2 being the target of fumagillin and ovalicin.
- p. 3 Results, 'Protac-1 Recruits MetAP-2 to SCF', end of first paragraph
shown; ref. 7)
- p. 5 Reference 7
Sin, N., Meng, L., Wang, M. Q., Wen, J. J., Bornmann, W. G. & Crews, C. M.
- p. 3 Results, 'Protac-1 Recruits MetAP-2 to SCF', first paragraph
manner that was inhibited by the IPP and Protac-1 (data not shown; ref. 7)
- p. 5 Reference 7
7. Sin, N., Meng, L., Wang, M. Q., Wen, J. J., Bornmann, W. G. & Crews, C. M.
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that Sin et al. 1997 concerns MetAP-2 as the target of fumagillin/ovalicin and not SCF or IκBα ubiquitination; the reference list prints no titles, so the content of ref. 7 is inferred from its authors and from the paper's own use of it in the Introduction ("fumagillin and ovalicin (OVA; refs. 7 and 8)"). Read the sentence on the page 3 image and the entry on page 5. The SCF assay in the same paragraph is attributed to ref. 13 (Tan et al., Mol. Cell 1999) and the IκBα phosphopeptide work to refs. 4 and 5, so one of those is the more likely intended source. The citation number looks wrong but only the authors can say which reference was meant.
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Reference or label Tissue-culture medium is described with "penicillin (100 units/ml), streptomycin (100 mg/ml)"; the unit for streptomycin is printed as mg/ml.
- p. 2 Methods, Tissue Culture
tomycin (100 mg
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: the usual cell-culture supplement is streptomycin at 100 µg/ml alongside penicillin at 100 units/ml; the paper itself gives no other value. The crop and page 2 image show "mg/ml" with a plain m, while µ is printed correctly two lines below in the same paragraph ("40 µg", "20 µg"), so this is not a font or extraction artefact. Probably µg/ml was intended.
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Reference or label The Fig. 4 legend has "(A) Ubiquitination of the 46-kDa fragment of MetAP-2", whereas the Methods describe "a 47-kDa fragment" and the Results "a 47-kDa autocatalyzed cleavage product (Fig. 4A)".
- p. 4 Fig. 4 legend (A)
the 46-kDa fragment of MetAP-2
- p. 3 Results, 'Protac-1 Mediates the Ubiquitination of MetAP-2'
one contained primarily a 47-kDa autocatalyzed cleavage prod
- p. 2 Methods, Immunoprecipitation and Ubiquitination Assays, last paragraph
47-kDa fragment that was generated
- p. 3 Results
47-kDa autocatalyzed cleavage prod-
Only the authors can say what is right here.
Judge's note: Read on the page 2, 3 and 4 images. The Methods say there were only two preparations (the fragment and full-length 67-kDa), and the Results tie the 47-kDa preparation to Fig. 4A, so the legend's 46 kDa is the same fragment. Two of three places say 47; the legend is probably the slip, but an apparent molecular weight is the authors' to state.
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Reference or label Results: "but not by other protease inhibitors (chymotrypsin, pepstatin, and leupeptin) added to the reaction"; the Methods name the inhibitors as "chymostatin, pepstatin, and leupeptin".
- p. 4 Results, 'MetAP-2–Protac-1 Is Degraded in Xenopus Egg Extracts'
by other protease inhibitors (chymotrypsin, pepstatin, and leu
- p. 2 Methods, Degradation Experiments with Xenopus Extracts
The protease inhibitors chymostatin, pepstatin, and
- p. 4 Results, Xenopus extracts
(chymotrypsin, pepstatin, and leu-
- p. 2 Methods
chymostatin, pepstatin, and
Judge's note: Read on the page 4 and page 2/3 images. The Results list is otherwise identical to the Methods list and is introduced as protease inhibitors; the Methods word is chymostatin. The internal mismatch is enough to confirm; that chymotrypsin is itself a protease rather than an inhibitor is outside knowledge that supports chymostatin as the intended word.
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Reference or label In the Fig. 4C lane table the "ubiquitin:" row reads Me, Me, Me, +, Me, +, − for lanes 1–7, so lane 7 is labelled as having neither ubiquitin nor methylubiquitin, yet the text uses it to show that "unlinked OVA (50 µM) plus IPP (50 µM) were not able to substitute for the OVA–IPP conjugate (lane 7)".
- p. 4 Fig. 4C, lane table, row 'ubiquitin:', lane 7
ubiquitin:
- p. 3 Results, last lines of page
able to substitute for the OVA–IPP conjugate (lane 7)
- p. 4 Fig. 4 legend (C)
methylubiquitin (Me) was substituted for ubiquitin, as
- p. 3 Results
were not able to substitute for the OVA–IPP conjugate (lane 7)
Only the authors can say what is right here.
Judge's note: Enlarged the panel from the PDF (5x render of page 4): the lane 7 cell in the ubiquitin row is a clear minus sign, distinct from the + in lanes 4 and 6 and the Me in lanes 1, 2, 3 and 5; lane 7 has + for SCF and + for IPP + OVA. The legend says only that methylubiquitin was substituted for ubiquitin "as indicated" and that lanes 5–7 lacked Protac-1; it does not say ubiquitin was left out of lane 7. As labelled, the lane could not show ubiquitination whatever was added. Either the label is a slip for + or Me, or the legend is incomplete; only the authors can say.
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Reference or label Reference 12 reads "Murray, A. W. (1991) Methods Cell Biol. 3, 581–605."; the volume number 3 appears to have lost a digit (the 1991 chapter is reported as volume 36).
- p. 5 Reference 12
Methods Cell Biol. 3, 581–605
- p. 5 Reference 12
Methods Cell Biol. 3, 581–605.
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that Murray's 1991 chapter "Cell cycle extracts" is in Methods in Cell Biology volume 36, pp. 581–605. The crop and page image show a bold "3," as the volume, so the quote is as printed and not an extraction artefact. Nothing in the files gives the true volume; a volume 3 of a long-running series dated 1991 is implausible, but the correct value should be checked against Crossref or the publisher.
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Reference or label Reference 16 reads "Liu, S., Widom, J., Kemp, C. W., Crews, C. M. & Clardy, J. (1998) Science 282, 324–327."; the page range appears to have lost its leading digit (reported as 1324–1327).
- p. 6 Reference 16
324–327
- p. 6 Reference 16
Science 282,
- p. 6 Reference 16, second line
324–327.
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that Liu et al. (1998), the structure of human MetAP-2 complexed with fumagillin, is at Science 282, 1324–1327. The crop shows "Science 282," followed on the next line by "324–327.", so the quote is as printed. The true page range cannot be verified from the files and should be checked against Crossref.
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Reference or label Reference 13 reads "(1999) Mol. Cell 3, 327–333."; the page range is reported to be 527–533. Separately, reference 20 abbreviates the journal as "Mol. Cell." where references 13 and 18 print "Mol. Cell".
- p. 6 Reference 13
(1999) Mol. Cell 3, 327–333.
- p. 6 Reference 20
Mol. Cell. 1, 565–574
- p. 6 Reference 13
Mol. Cell 3, 327–333
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that Tan et al. (1999) is at Mol. Cell 3, 527–533. The crop shows "Mol. Cell 3, 327–333." as printed; the true range cannot be verified from the files and should be checked against Crossref. The second point is visible in the files: ref. 20 prints "Mol. Cell. 1, 565–574" with a full stop after "Cell", while refs 13 and 18 print "Mol. Cell" without one; that is a cosmetic (S4) style inconsistency.
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10 minor (typos and the like; most checked by the judge only)
Wording Fig. 1B defines R as "GGGGGGRAEDS*GNES*EGE-COOH or GGGGGGDRHDS*GLDS*M-COOH"; only the second contains the IκBα peptide "DRHDSGLDSM" the paper describes, and the first sequence and the six-glycine spacer are not mentioned in the text, Methods or legend.
- p. 2 Fig. 1B, definition of R under the final structure
R = GGGGGGRAEDS*GNES*EGE-COOH
- p. 1 Introduction
peptide within IkBa, DRHDSGLDSM (4, 5)
Only the authors can say what is right here.
Judge's note: Enlarged Fig. 1B from the PDF (5x render of page 2): the label under the final structure gives two alternative sequences for R, with "* phosphorylated serine". Searched the text: the only peptide described is the 10-aa IκBα peptide DRHDSGLDSM (Introduction); Methods say only "IκBα peptide"; the legend says "The synthesis scheme for Protac-1". The RAEDS*GNES*EGE sequence is never identified (the Discussion mentions a Vpu-derived β-TRCP-binding peptide only as a future option, without a sequence). The figure shows a compound variant the paper does not explain; not an error in the reported data.
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Wording Methods say the 47-kDa MetAP-2 fragment "was generated either by a contaminating protease or by slow autoproteolysis", but the Results call it without qualification "a 47-kDa autocatalyzed cleavage product".
- p. 2 Methods, Immunoprecipitation and Ubiquitination Assays, last paragraph
generated either by a contaminating
- p. 3 Results, 'Protac-1 Mediates the Ubiquitination of MetAP-2'
47-kDa autocatalyzed cleavage prod
Judge's note: Both sentences read on the page 2 and page 3 images. The Methods leave the origin open between two causes; the Results assert one of them. A wording inconsistency with no effect on the data.
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Wording The Methods recipe for ubiquitination reactions ("4 µl of 18 µM MetAP-2, 4 µl of 100 µM Protac-1, 0.5 µl of 0.1 µg/µl purified mouse E1, 1 µl of 0.5 µg/µl human Cdc34 E2, and 1 µl of 25 mM ATP were added to 20 µl ... beads") lists no ubiquitin or methylubiquitin, although the Results and Fig. 4 legend say ubiquitin was added and Fig. 4B varies "E1, E2, plus ubiquitin" at 1X and 2X.
- p. 2 Methods, Immunoprecipitation and Ubiquitination Assays
of 25 mM ATP were added to 20
- p. 3 Results, 'Protac-1 Mediates the Ubiquitination of MetAP-2'
ATP, MetAP-2–Protac-1, plus purified E1, E2 (human Cdc34),
Only the authors can say what is right here.
Judge's note: Read the Methods paragraph on the page 2 image and the Results/legend on pages 3 and 4. Searched the whole text for an amount or source of ubiquitin or methylubiquitin: none is given anywhere. The omission is real; the amount of ubiquitin cannot be recovered from the paper. An incomplete methods description rather than a wrong number.
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Wording The Fig. 3 control is described in the Results as "control beads preincubated with untransfected 293T cell lysates", but in the Fig. 3 legend as extracts from cells "transiently transfected with either control vector or plasmids expressing FLAG epitope-tagged Cul-1 and β-TRCP" and in the Methods as "Pellets from cells transfected with vector".
- p. 3 Results, 'Protac-1 Recruits MetAP-2 to SCF', second paragraph
preincubated with untransfected 293T cell lysates
- p. 3 Fig. 3 legend
transiently transfected with either control vector or plasmids expressing FLAG
- p. 2 Methods, Immunoprecipitation and Ubiquitination Assays
Pellets from cells transfected with vector, pFLAG-
Judge's note: Read all three on the page 2 and page 3 images. Untransfected and vector-transfected cells are different controls as written; the legend and Methods agree with each other. Possibly loose wording for "not transfected with the SCF plasmids". Does not change the result shown.
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Typo The abstract expands Protac as "protein-targeting chimeric molecule 1 (Protac-1)", whereas the Introduction has "proteolysis-targeting chimeric molecule 1 (Protac-1)" and the abbreviations footnote has "Protac, proteolysis targeting chimeric".
- p. 1 Abstract
protein-targeting chimeric molecule 1 (Protac-1)
- p. 1 Introduction, last paragraph
proteolysis-targeting chimeric molecule 1 (Protac-1)
- p. 1 Abbreviations footnote
Protac, proteolysis targeting chimeric
- p. 1 Introduction
proteolysis-targeting chimeric
Judge's note: Read all three places on the page 1 image. The abstract line break reads "protein-" / "targeting chimeric molecule 1 (Protac-1)"; the Introduction and the footnote both say proteolysis. One abbreviation, two expansions on one page; two of the three agree, so the abstract is the odd one out. Nothing in the paper presents the two as deliberate alternatives.
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Typo Reference 2 reads "Winston, J. F., Koepp, D. M. Zhu, C., Elledge, S. J. & Harper, J. W."; the comma between "Koepp, D. M." and "Zhu, C." is missing.
- p. 5 Reference 2
Koepp, D. M. Zhu, C.
- p. 5 Reference 2
Koepp, D. M. Zhu, C., Elledge
Judge's note: Checked on the crop: "Koepp, D. M. Zhu, C." is printed with no comma after "M.", while every other author in the list is separated by a comma. Both names sit on one line, so it is not a line-break artefact.
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Typo The acknowledgements thank "Zhen-Quiang Pan"; the given name appears to be misspelled ("Zhen-Qiang"), the same person being cited in reference 13 as "Pan, Z.-Q.".
- p. 5 Acknowledgements
Zhen-Quiang Pan
- p. 6 Reference 13
Pan, Z.-Q.
- p. 6 Reference 13
Ronai, Z. & Pan, Z.-Q.
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that the given name of Z.-Q. Pan (senior author of ref. 13) is spelled Zhen-Qiang. The crop shows "Zhen-Quiang Pan" as printed. Reference 13 gives only the initials "Z.-Q.", which fit either spelling, so the files do not settle it; "Quiang" is not a standard pinyin syllable, which makes a misspelling likely.
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Typo Reference 19 reads "Snyder, E. L., Ho., A., Latham, D. G."; there is a stray full stop after the surname "Ho".
- p. 6 Reference 19
Ho., A.
- p. 6 Reference 19
Ho., A., Latham, D. G.
Judge's note: The crop shows "Ho., A." with a full stop between the surname and the comma. Every other entry uses "Surname, Initials"; ref. 10 on the previous page prints "Ho, S. N." without a stop.
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Typo Reference 18 reads "Zhou, P., Bogachi, R., McReynolds, L. & Howley, P. M. (2000) Mol. Cell 6, 751–756."; the second author's surname appears to be misspelled (reported as Bogacki).
- p. 6 Reference 18
Zhou, P., Bogachi, R.
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that the second author of Zhou et al. (2000) Mol. Cell 6, 751–756 is R. Bogacki. The crop shows "Bogachi, R." as printed. The name appears nowhere else in the article, so the files cannot settle the spelling.
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Typo Reference 14 reads "Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennet, B. L., Li, J. W."; the surname "Bennet" appears to be misspelled (reported as Bennett).
- p. 6 Reference 14
Bennet, B. L.
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that the fifth author of Mercurio et al. (1997) Science 278, 860–866 is B. L. Bennett. The crop shows "Bennet, B. L." as printed. The name appears nowhere else in the article, so the files cannot settle the spelling.
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