Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt

Pratt, Matthew R.; Schwartz, Edmund C.; Muir, Tom W.

doi:10.1073/pnas.0700816104

Cited by 89 publications

(88 citation statements)

References 36 publications

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“…Finally, it is important to note that this method differs significantly to previous applications of FKBP and FRAP to regulate protein function-for example, dimerization to initiate signaling pathways (35) or transcription (36), destabilized FKBP (37) or split ubiquitin (38) to affect protein degradation, or regulation of an inhibitory peptide (39). Specifically, the applications invariably tether FKBP and/or FRAP to the protein of interest and must express this fusion in a background containing the protein of interest or in cells where the protein of interest has been suppressed or deleted.…”

Section: Discussionmentioning

confidence: 99%

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes

Varma

Dawn

Ghosh-Roy

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dyneinmediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.in vivo antagonist | retrograde transport | organelle kinetics | mitotic index C ytoplasmic dynein is a microtubule-based motor protein involved in numerous essential cellular functions, including intracellular transport of membranous organelles and macromolecular complexes, mitosis, and cell migration (1). Cytoplasmic dynein consists of multiple subunits: two 532-kDa heavy chains (HCs), each containing a motor domain, and a number of accessory subunits, the intermediate, light intermediate, and light chains (ICs, LICs, and LCs, respectively). The ICs and LCs form a complex at the base of the dynein HC (residues 1-1,100) (2). The ICs have been implicated in subcellular targeting of cytoplasmic dynein through an interaction with a large accessory complex, dynactin (3-6). The LCs, in turn, form a complex with the dynein IC, but also interact with a large number of other proteins, including transcription factors, signaling molecules, RNA, and viral proteins. As such, the dynein LCs have been implicated as adaptors to link these diverse molecules to dynein for retrograde transport (1).Our recent structural and biophysical studies aimed at understanding role of the dynein LCs, however, do not support the direct involvement of these subunits in cargo recognition and transport (7). Specifically, the structure of the LCs [LC8 (Dynll1) and TcTex1 (Dynlt1)] bound to the dynein IC shows that the dynein IC occupies ...

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Section: Discussionmentioning

confidence: 99%

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes

Varma

Dawn

Ghosh-Roy

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…SURF technology was recently developed by Pratt et al (19) and makes use of the previously described degron approach with an additional feature enabling release of the native protein from the degron sequence upon small molecule-induced rescue of the protein-degron chimera from degradation. The authors split ubiquitin into N-terminal UbN(I13A) (residues 1-37) and C-terminal UbC (residues 35-76) fragments.…”

Section: Surf Technologymentioning

confidence: 99%

Chemical Inducers of Targeted Protein Degradation

Raina

Crews

2010

Journal of Biological Chemistry

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“…Muir and co-workers have recently reported a CID-based system that is, in principle, even more generalizable in that it does not require that localization or multimerization of a particular POI is inherently activating [52]. In this approach, the addition of a CID produces a dimerization event that rescues the target protein from proteolysis.…”

Section: Enzyme Activation Using Chemical Inducers Of Dimerizationmentioning

confidence: 99%

Brought to life: targeted activation of enzyme function with small molecules

Bishop

Chen

2008

J Chem Biol

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Cell-permeable small molecules that are capable of activating particular enzymes would be invaluable tools for studying protein function in complex cell-signaling cascades. But, is it feasible to identify compounds that allow chemical-biology researchers to activate specific enzymes in a cellular context? In this review, we describe some recent advances in achieving targeted enzyme activation with small molecules. In addition to surveying progress in the identification and targeting of enzymes that contain natural allosteric-activation sites, we focus on recently developed protein-engineering strategies that allow researchers to render an enzyme of interest "activatable" by a pre-chosen compound. Three distinct strategies for targeting an engineered enzyme are discussed: direct chemical "rescue" of an intentionally inactivated enzyme, activation of an enzyme by targeting a de novo smallmolecule-binding site, and the generation of activatable enzymes via fusion of target enzymes to previously characterized small-molecule-binding domains.

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Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt

Cited by 89 publications

References 36 publications

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes

Chemical Inducers of Targeted Protein Degradation

Brought to life: targeted activation of enzyme function with small molecules

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