REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING COMPACT DISK APPENDIX

The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 27, 2018, is named UCSF050PCT_SL.txt and is 7,765 bytes in size.

BACKGROUND OF THE INVENTION

The monitoring of protein-protein interactions in living cells is a vital endeavor in biological and medical research. Various research tools have been developed for the study of such interaction in vivo, such as Forster resonance energy transfer (FRET) reporters and bimolecular fluorescence complementation assays. However, the tools developed to date suffer from the complications of imaging dispersed fluorescent proteins in living cells and animals. Signal measurement and interpretation is challenging with current assays due to low fluorescence changes, complex signal patterns, tissue autofluorescence, light scattering, and rapid cell movement and shape changes.

Meanwhile, in natural systems, phase-separated droplets formed by multivalent proteins create distinct structures within the cell, with unique microenvironments that can facilitate certain enzymatic activities. The prevalence and importance of these phase-separated bodies in biology has only recently been appreciated. In a recapitulation of these natural systems, Li et al., (2012), Phase transitions in the assembly of multivalent signaling proteins. Nature 483, 336-340, constructed artificial multivalent reporters of protein-protein interactions, comprising reporter proteins functionalized with multiple copies of two interacting proteins. When the protein-protein interactions were induced, the reporter proteins aggregated, creating localized phase separated bodies in living cells that were readily observed.

Li's work demonstrated the potential of synthetic multivalent proteins as indicators of protein-protein interactions. However, Li's reporter proteins comprised large, bulky proteins having several repeating protein domains. Applying the reporter system of Li is problematic because the efficient expression of such large proteins is difficult, and the size of the reporter constructs can hinder the protein-protein interactions to be studied. Furthermore, the repeating domains used in this prior method are problematic for making transgenic animals due to the high potential for DNA recombination.

Accordingly, there remains a need in the art for superior assays for probing protein-protein interactions. There is a need for a more efficient, customizable platform for reporting protein-protein interactions. There is a need for more robust fluorescent reporters for detecting and imaging protein-protein interactions. There is a need for detecting and imaging these protein-protein interactions in various situations, including in vivo imaging within living animals. There is a need for a fluorescent protein-protein interaction reporter that has a large signal-to-noise ratio and fast kinetics. There is a need for novel synthetic multivalent proteins which can act as indicators of selected protein-protein interactions by the formation of phase-separated intracellular droplets, wherein such reporters are efficiently expressed and wherein such systems are highly customizable and effective.

SUMMARY OF THE INVENTION

The various inventions disclosed herein fulfill the aforementioned needs in the art. The present invention encompasses novel reporters of protein-protein interactions. The overall operation and objective of the invention encompasses the use of two functionalized orthogonal monomers, each one comprising one partner of a protein-protein interaction. The monomers can be readily expressed together in cells wherein homo-oligomers of the two monomer types will form. Upon the occurrence of the protein-protein interaction, the homo-oligomers will cross-link by the binding of the interacting protein partners. The result is a localized network of highly condensed oligomers which results in phase shifting and the segregation of bright signal in distinct punctae.

In a primary implementation, the invention employs compact, coiled coil based monomers. In the practice of the invention, two different types of coiled coil oligomeric monomers are made, each type functionalized with complementary partners of a targeted protein-protein interaction, and being further functionalized with at least one fluorescent protein. In living cells, expression of the reporter proteins results in the spontaneous formation of oligomeric reporter assemblies, each comprising several copies of one of the interacting proteins and at least one of the pair comprising several copies of a fluorescent protein reporter. When the protein-protein interaction is activated, the interacting proteins of each oligomer will be connected to multiple copies of the complementary oligomer by the interactions of the complementary proteins. Multivalent complexes are formed that concentrate the fluorescent labels in phase-separated intracellular droplets. These localized and intense punctae are readily visualized and quantified. The kinetics of the multivalent complex formation are fast and efficient. The reporter proteins may be readily expressed and imaged in cells and living organisms.

The novel inventions of the present disclosure improve upon the prior art by the use of coiled coil subunits as the backbone of the oligomeric reporter elements. As set forth below, the use of coiled coil proteins has numerous advantages, providing the art with an effective and versatile system for monitoring protein-protein interactions.

In one aspect, the scope of the invention encompasses novel reporter constructs comprising coiled coil proteins which may be customized and used to detect a selected protein-protein interaction. In the presence of the reporter system, the selected interaction creates multivalent complexes and readily visualized signals in phase-shifted droplets.

In one aspect, the scope of the invention encompasses novel genetic constructs and engineered cells and organisms which express the reporter constructs of the invention.

In one aspect, the scope of the invention encompasses methods of observing and/or quantifying a target protein-protein interaction and provides methods for elucidating the factors that modulate the target protein-protein interaction or testing modulators of the selected interaction.

In one aspect, the scope of the invention encompasses novel tools for measuring the activity of enzymes, wherein the protein-protein interaction are driven by the activity of the enzyme.

In one embodiment, the invention encompasses novel tools for detecting kinase activity. Kinase-substrate interactions serve as major control points for innumerable biological processes. In certain implementations, the constructs of the invention can be used to monitor kinase interactions with substrates.

In one aspect, the scope of the invention encompasses novel tools for measuring the activity of proteolysis targeting chimera (PROTAC) constructs, which are chimeric molecules designed to specifically degrade target proteins. The reporters of the invention encompass constructs and methods for evaluating and improving PROTACs and like tools.

