Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation

Abstract

The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, protein-targeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the I kappa B alpha phosphopeptide that is recognized by the F-box protein beta-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCF(beta-TRCP), ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.

Figure 1

(A) Protac-1 targets MetAP-2 to SCF. Protac-1 is a chimeric molecule that consists of a phosphopeptide moiety and a small molecule moiety that interacts with the protein target. Ub, ubiquitin; H, Hrt1. (B) The synthesis scheme for Protac-1 (see Materials and Methods). Fmoc, fluorenylmethoxycarbonyl; DMF, dimethylformamide; DMAP, dimethylaminopyridine; DSS, disuccinimidyl suberate.

Figure 2

MetAP-2 binds Protac specifically and in a concentration-dependent manner. (A) MetAP-2 (9 μM) was incubated with increasing concentrations of Protac-1 at room temperature for 45 min. The last two lanes depict MetAP-2 that was incubated with either free IPP (50 μM) or free OVA (50 μM), as indicated. After incubation, samples were supplemented with SDS/PAGE loading buffer, separated by SDS/PAGE, and immunoblotted with MetAP-2 antiserum. (B) Same as A, except MetAP-2 (9 μM) plus Protac-1 (10 μM) were supplemented with either IPP (50 μM) or OVA (50 μM), as indicated. Protac binding to MetAP-2 was inhibited by the addition of OVA, but not by the addition of IPP.

Figure 3

Protac-1 recruits MetAP-2 to SCF^β-TRCP. Extracts from 293T cells transiently transfected with either control vector or plasmids expressing FLAG epitope-tagged Cul-1 and β-TRCP were subject to affinity purification on anti-FLAG resin to yield either control beads or SCF^β-TRCP beads. The matrices then were mixed with the preformed MetAP-2–Protac-1 complex (input), incubated, and separated into pellet (bound) and unbound (sup) fractions. Proteins were fractionated on an SDS/10% polyacrylamide gel, and immunoblotted with anti-MetAP-2 antiserum. MetAP-2 and MetAP-2–Protac-1 refer to free MetAP-2 and MetAP-2 complexed with Protac-1, respectively.

Figure 4

Protac mediates MetAP-2 ubiquitination by SCF. (A) Ubiquitination of the 46-kDa fragment of MetAP-2. MetAP-2–Protac-1 mixture was added to either control (Mock) or SCF^β-TRCP beads (+) supplemented with ATP plus purified E1, E2 (Cdc34), and ubiquitin. The E2, UbcH5c (500 ng), was also tested in the reaction, which resulted in the same degree of ubiquitination as observed with Cdc34 (data not shown). Reactions were incubated for 1 hr at 30°C and were evaluated by SDS/PAGE followed by Western blotting with anti-MetAP-2 antiserum. (B) Ubiquitination of full-length (67-kDa) MetAP-2. Same as A, except that the 67-kDa preparation of MetAP-2 was used, and E1, E2, plus ubiquitin were either added at normal (1X) or 2-fold higher (2X) levels, as indicated. (C) Ubiquitination of MetAP-2 by SCF^β-TRCP depends on Protac-1. Same as A, except that methylubiquitin (Me) was substituted for ubiquitin, as indicated, and the reactions depicted in lanes 5–7 lacked Protac-1. In lane 7, unlinked IPP and OVA were added at 100 μM in place of Protac-1. (D) Protac-1-dependent ubiquitination of MetAP-2 is competitively inhibited by IPP. Same as A, except that reactions in lanes 3 and 4 were supplemented with 100 μM each IPP and OVA, respectively.

Figure 5

MetAP-2-Protac is degraded in Xenopus extracts. The MetAP-2–Protac-1 mixture or MetAP-2 alone was added to Xenopus egg extract fortified with OVA (100 μM), IKK-EE (0.4 μg), and OA (10 μM). Where indicated, reactions were either deprived of IKK-EE or OA, or were further supplemented with 50 μM LLnL or 10 μM epoxomicin (Epox). Reactions were incubated for the indicated times at room temperature, terminated by adding SDS/PAGE loading buffer, and evaluated by SDS/PAGE followed by Western blotting with anti-MetAP-2 antiserum.

Figure 6

General application of Protacs. A schematic of how different disease-promoting proteins might be recruited to different ubiquitin ligases for ubiquitination and degradation by unique Protacs.