Mindy I. Davis Ph.D. National Institute of Health
Researchers describe a system for the rapid and complete degradation of proteins in mammalian cells.
The ability to rapidly and reversibly control protein levels in a cell can be important to determine gene function and to assist with target validation. Methods, such as RNA interference (RNAi), can be slow to deplete protein levels, particularly for long-lived proteins, such as CENP-A (centromere protein A). The authors* describe a method that takes advantage of a plant degradation system in which plant TIR1 is inducibly expressed in the cell type of interest where it can bind to the endogenous Skp1, Cullin, and F-box protein (SCF) ubiquitin ligase complex (see Figures 1A, B, C, and D).
Figures 1A–B. Auxin-inducible degradation system for controlling protein stability in human cells. (A) Schematic illustration of the SCF ubiquitin ligase. The variable F-box protein is involved in substrate recruitment. (B) Schematic illustration of the auxin-induced degradation system. Ectopically expressed TIR1 F-box protein incorporates into the SCF complex in human cells. In the presence of the IAA, TIR1 associates with the AID fused to the protein of interest (POI). SCFTIR1 recruits an E2 ligase and polyubiquitinates the AID, resulting in the degradation of the POI by the proteasome.
When the protein of interest, which needs to be tagged with auxin-inducible degron (AID), is co-expressed in these cells, it can be bound by the TIR1-SCF complex; binding induces ubiquitination and degradation of the tagged protein. It is important to note that the endogenous protein levels are not affected by this technique. Therefore, depending on the desired application, this technique may need to be combined with small interfering RNA (siRNA) for the endogenous protein or the AID tag may need to be added to the endogenous protein through genome engineering.
Figures 1C–D. (C) Parental and TIR1-9Myc expressing DLD-1 (a monoclonal colorectal cancer cell line) and RPE1 (a diploid non-tumor cell line) were treated with or without IAA for 3 d and processed for flow cytometry. Note expression of TIR1-9Myc and/or treatment with IAA does not cause alterations in the cell cycle profile. (D) DLD-1 or RPE1 cells stably expressing TIR1-9Myc were cotransfected with histone H2BmRFP or histone H2BAID-YFP; after 24 h, they were treated with (+) or without (-) IAA for a further 24 h. Fluorescent images show the presence or absence of histone H2BmRFP and histone H2BAID-YFP. (Scale bars = 5 µm.) SCF, Skp1, Cullin, and F-box protein; IAA, indole-3-acetic acid; AID, auxin-inducible degron; YFP, yellow fluorescent protein.
Figures 1C–D
This method was tested on three proteins that are nuclear and found in protein complexes (histone H2B, telomere repeat-binding factor 2 [TRF2], and CENP-A) and two that are cytoplasmic (cyclin B1 and polo-like kinase 4 [PLK4]). The proteins have a rapid t1/2 (average of 19 minutes) with quantitative loss within 80 minutes, and the degradation was inhibited by the proteasome inhibitor MG132 (see Figure 2). Even the usually long-lived centromere-bound protein CENP-A was rapidly degraded by this technique.
Additionally, for histone H2B, it was shown that the degradation was efficient in all cell cycle phases. The effect was shown to be reversible, and for PLK4 new protein was detected within just 10 minutes of washout of the TIR1 inducer indole-3-acetic acid (IAA). In contrast, RNAi is not readily reversible.
The authors point out that it is not yet clear if this method can be used in animals due to possible toxicity of the IAA induction system. An alternate approach to reduce the levels of tagged protein in cells, called the HaloTag approach, has been shown previously to be effective in mice and in zebrafish embryos (Neklesa et al., Nat Chem Biol 2011;7:538–543.). This approach involves expressing a protein fused to a bacterial dehalogenase protein (i.e., the HaloTag), and then a small molecule that binds to this tag is added. The small molecule contains a large hydrophobic motif, such as adamantyl, that mimics the partially denatured state of a protein and leads to the degradation of the protein.
For a protein for which a small molecule cell-permeable inhibitor exists, it will be interesting to compare the various techniques (RNAi, HaloTag, small molecule inhibition) to the AID method described in this article because it is possible that the phenotypes could differ depending on how rapidly and completely the protein of interest can be depleted or inactivated.
Figure 2. Protein degradation occurs rapidly following IAA addition. AID-YFP-tagged proteins were induced for 8–24 h, and cells were treated with IAA in the presence or absence of the proteasome inhibitor MG132. EYFP levels were monitored by fluorescence time-lapse microscopy beginning 9–10 min after drug addition. (A–D) Graphs show EYFP fluorescence at various times after drug addition. (E–H) Graphs show the logarithmic plot of EYFP fluorescence at various time points after filming began. Graphs represent the mean of 7–20 cells per condition. Error bars represent the SEM. EYFP, enhanced yellow fluorescent protein.
*Abstract from PNAS USA 2012, Vol. 109: E3350–E3357
Inducible degradation is a powerful approach for identifying the function of a specific protein or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to degrade AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AID-tagged protein can functionally replace an endogenous protein depleted by RNAi, leading to an inducible null phenotype rapidly after auxin addition.
The AID system is shown to be capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 min and proceeding to completion with first-order kinetics. AID-mediated instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue in all cell cycle phases, including mitosis, making this system especially useful for identifying the function(s) of proteins of interest during specific points in the mammalian cell cycle.
Mindy I. Davis, Ph.D., works at the NIH.
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Proceedings of the National Academy of Sciences USA titled “Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells.” Authors of the paper are Holland AJ, Fachinetti D, Hana JS, and Cleveland DW.
