Patent Grant
10324085
The present invention relates to the real-time detection of protein-protein interactions, and more particularly multiprotein complexes, e.g., ternary or quaternary complexes, in living cells.
Pursuant to 37 C.F.R. 1.821(c), the sequence listing submitted on Jun. 22, 2016 as an ASCII compliant text file named 15691_67-seq_listing_ST25.txt, created on Dec. 23, 2014 and having a size of ˜14 kilobytes, is hereby incorporated by reference in its entirety.
Fluorescence/Förster Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) both allow real-time detection of protein-protein interactions in intact cells. FRET involves energy transfer between two fluorophores (fluorescent proteins). A donor fluorophore, initially in its electronic excited state, may transfer energy to an acceptor fluorophore through nonradiative dipole-dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor making FRET extremely sensitive to small distances. Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other. A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise (autofluorescence, light scattering and/or photoisomerization) due to direct excitation of the acceptor or to photobleaching. Photobleaching is an important drawback when assessing endogenous interactions because it can damage the cells and therefore alter normal interactions between biomolecules.
To avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. BRET assay technology is based on the efficient resonance energy transfer (RET) between a bioluminescent donor moiety and a fluorescent acceptor moiety. This technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than a fluorophore (typically Cyan fluorescent protein (CFP)) to produce an initial photon emission compatible with the fluorescent acceptor (typically yellow fluorescent protein (YFP)).
FRET allows reliable monitoring of sequential transfer between three fluorophores in a three-color or triple-FRET assay, but still suffers from photobleaching of the donor and contaminating cross-excitation problems. Sequential Resonance Energy Transfer (SRET), which combines serially BRET between an initial donor and intermediate acceptor and resonance energy transfer between the intermediate acceptor and a terminal acceptor (
As many biomolecules interact in ternary (or higher order complexes), there remains a need for assays that allow for reliable ternary/quaternary complex monitoring.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
Described herein is a bioluminescence resonance energy transfer (BRET) based system, called BRET with Fluorescence enhancement by combined energy transfer (BRETFect), to monitor ternary complex formation in real-time in live cells with high sensitivity and accuracy. As opposed to SRET (in which the energy is transferred from an initial donor (Luc) to an intermediate acceptor (GFP), which in turn transfers the energy to the terminal acceptor (YFP), see
The present invention provides the following items 1 to 54.
1. A biosensor for detecting a ternary protein complex comprising: i. a first protein tagged with a Donor (D) protein, wherein D is a bioluminescent protein (Biol) having an emission spectrum (Biol-Em); ii. a second protein tagged with an Intermediate (I) protein, wherein I is a first Fluorescent protein (FP1) having an excitation spectrum (FP1-Ex) and emission spectrum (FP1-Em); and iii. a third protein tagged with an Acceptor (A) protein, wherein A is a second fluorescent protein (FP2) having an excitation spectrum (FP2-Ex) and an emission spectrum (FP2-Em); wherein a) the FP1-Ex overlaps with the Biol-Em and overlaps minimally with the FP2-Ex; b) the FP1-Em overlaps with the FP2-Ex; c) the FP2-Ex overlaps with the Biol-Em; and d) the FP2-Em has a longer wavelength than the FP1-Em.
2. A biosensor for the detection of a quaternary protein complex comprising: i. a first protein tagged with a first portion of a Donor protein (D1), wherein D1 is a first bioluminescent protein portion (BiolP1), ii. a second protein tagged with a second portion of a Donor protein (D2), wherein D2 is a second bioluminescent protein portion (BiolP2), wherein interaction between said first and second proteins brings said BiolP1 and BiolP2 in close enough proximity to form a functional bioluminescent protein (Biol) having an emission spectrum Em; iii. a third protein tagged with an Intermediate (I) protein, wherein I is a first Fluorescent protein (FP1) having an excitation spectrum (FP1-Ex) and emission spectrum (FP1-Em); and iv. a fourth protein tagged with an Acceptor (A) protein, wherein A is a second fluorescent protein (FP2) having an excitation spectrum (FP2-Ex) and an emission spectrum (FP2-Em); wherein a) the FP1-Ex overlaps with the Biol-Em and overlaps minimally with the FP2-Ex; b) the FP1-Em overlaps with the FP2-Ex; c) The FP2-Ex overlaps with the Biol-Em; and d) the FP2-Em has a longer wavelength than the FP1-Em.
3. The biosensor of item 1 or 2, wherein the bioluminescent protein is a luciferase, preferably a Renilla Luciferase (RLuc).
4. The biosensor of any one of items 1-3, wherein FP1 is mTFP1 or mTagBFP2 fluorescent protein.
5. The biosensor of any one of items 1-4, wherein FP2 is Venus, Topaz or mTFP1 fluorescent protein.
6. The biosensor of any one of items 1-5, further comprising a luciferase substrate.
7. The biosensor of item 6, wherein the bioluminescent protein substrate is a coelenterazine.
8. The biosensor of item 7, wherein the coelenterazine is coelenterazine H or coelenterazine-400a.
9. The biosensor of any one of items 1-8, wherein said first, second, third or fourth protein is a nuclear receptor protein or a fragment thereof.
10. The biosensor of item 9, wherein at least two of said first, second, third and/or fourth proteins are nuclear receptor proteins or fragments thereof.
11. The biosensor of item 10, wherein two of said first, second, third and/or fourth proteins are nuclear receptor proteins or fragments thereof.
12. The biosensor of item 11, wherein said two nuclear receptor proteins or fragments thereof are identical.
13. The biosensor of item 11, wherein said two nuclear receptor proteins or fragments thereof are different.
14. The biosensor of any one of items 9 to 13, wherein the nuclear receptor is an Estrogen receptor (ER), a Retinoic acid receptor, Androgen receptor (AR), Glucocorticoid receptor (GR) or Progesterone receptor (PR).
15. The biosensor of item 14, wherein the nuclear receptor is a nuclear estrogen receptor or a retinoic acid receptor.
16. The biosensor of any one of items 9 to 15, wherein at least one of said first, second, third or fourth protein is a nuclear receptor coactivator protein or a nuclear receptor-binding fragment thereof.
17. The biosensor of item 16, wherein said nuclear receptor coactivator protein is SRC1 or SRC2.
18. The biosensor of any one of items 1-8, wherein at least one of said first, second, third or fourth protein is a G protein coupled receptor (GPCR) protein or a fragment thereof.
19. The biosensor of item 18, wherein at least two of said first, second, third and/or fourth proteins are GPCR proteins or fragments thereof.
20. The biosensor of item 19, wherein two of said first, second, third and/or fourth proteins are GPCR proteins or fragments thereof.
21. The biosensor of item 20, wherein said two GPCR proteins or fragments thereof are identical.
22. The biosensor of item 20, wherein said two GPCR proteins or fragments thereof are different.
23. The biosensor of any one of items 18-22, wherein at least one of said first, second, third or fourth protein is a G protein subunit or a fragment thereof.
24. The biosensor of any one of items 18-22, wherein two of said first, second, third and/or fourth proteins are two different G protein subunits or fragments thereof.
25. The biosensor of item 23 or 24, wherein said G protein subunit(s) is/are a Gα subunit, a Gγ subunit and/or a Gβ subunit.
26. The biosensor of any one of items 18-23, wherein at least one of said first, second, third or fourth protein is a βarrestin protein or a fragment thereof.
27. The biosensor of any one of items 18-26, wherein at least one of said first, second, third or fourth protein is a pleckstrin homology (PH) domain-containing protein.
28. The biosensor of item 27, wherein said PH domain-containing protein is a G protein-coupled receptor kinase (GRK) protein or a PH domain of a GRK.
29. The biosensor of any one of items 18-23, wherein at least one of said first, second, third or fourth protein is a regulator of G-protein signalling (RGS), an activator of G-protein signalling (AGS), or a resistance to inhibitors of cholinesterase 8 protein (Ric-8), or any fragment thereof.
30. The biosensor of any one of items 1-29, further comprising a cell expressing components (i), (ii) and (iii).
31. A method for the detection of a ternary protein complex, the method comprising:
A) providing: i. a first protein tagged with a Donor (D) protein, wherein D is a bioluminescent protein (Biol) having an emission spectrum (Biol-Em); ii. a second protein tagged with an Intermediate (I) protein, wherein I is a first Fluorescent protein (FP1) having an excitation spectrum (FP1-Ex) and emission spectrum (FP1-Em); and iii. a third protein tagged with an Acceptor (A) protein, wherein A is a second fluorescent protein (FP2) having an excitation spectrum (FP2-Ex) and an emission spectrum (FP2-Em);
wherein a) the FP1-Ex overlaps with the Biol-Em and overlaps minimally with the FP2-Ex; b) the FP1-Em overlaps with the FP2-Ex; c) the FP2-Ex overlaps with the Biol-Em; and d) the FP2-Em has a longer wavelength than the FP1-Em; and
B) contacting the D protein with a bioluminescent protein substrate; and
C) detecting Bioluminescence Resonance Energy Transfer (BRET) with Fluorescence enhancement by combined energy transfer (BRETFect) signal; wherein the detection of a BRETFect signal is indicative that a complex is formed.
32. A method for the detection of a quaternary protein complex, the method comprising:
A) providing: i. a first protein tagged with a first portion of a Donor protein (D1), wherein D1 is a first bioluminescent protein portion (BiolP1) ii. a second protein tagged with a second portion of a Donor protein (D2), wherein D2 is a second bioluminescent protein portion (BiolP2), wherein interaction between said first and second proteins brings said BiolP1 and BiolP2 in close enough proximity to form a functional bioluminescent protein (Biol) having an emission spectrum Em; iii. a third protein tagged with an Intermediate (I) protein, wherein I is a first Fluorescent protein (FP1) having an excitation spectrum (FP1-Ex) and emission spectrum (FP1-Em); and iv. a fourth protein tagged with an Acceptor (A) protein, wherein A is a second fluorescent protein (FP2) having an excitation spectrum (FP2-Ex) and an emission spectrum (FP2-Em);
wherein a) the FP1-Ex overlaps with the Biol-Em and overlaps minimally with the FP2-Ex; b) the FP1-Em overlaps with the FP2-Ex; c) The FP2-Ex overlaps with the Biol-Em; and d) the FP2-Em has a longer wavelength than the FP1-Em; and
B) contacting the D1 and/or D2 protein with a bioluminescent protein substrate; and
C) detecting Bioluminescence Resonance Energy Transfer Forster enhanced by simultaneous transfer (BRETFect) signal; wherein the detection of a BRETFect signal is indicative that a quaternary complex is formed.
33. The method of item 31 or 32, wherein the bioluminescent protein is a luciferase, preferably a Renilla Luciferase (RLuc).
34. The method of any one of item 31-33, wherein FP1 is mTFP1 or mTagBFP2 fluorescent protein.
35. The method of any one of items 31-34, wherein FP2 is Venus, Topaz or mTFP1 fluorescent protein.
36. The method of any one of items 31-35, wherein the bioluminescent protein substrate is a coelenterazine.
37. The method of item 36, wherein the coelenterazine is coelenterazine H or coelenterazine-400a.
38. The method of any one of items 31-37, wherein the detecting step in C) comprises detecting the D emission at about 485 nm and the A emission at between about 530 and 550 nm.
39. The method of item 38, wherein the A emission is detected at about 550 nm.
40. The method of any one of items 31-39, wherein said first, second, third and fourth proteins are as defined in any one of items 9 to 29.
41. A method for determining whether an agent modulates the formation of a ternary or quaternary protein complex comprising performing the method of any one of items 31-40 in the presence and in the absence of said agent, wherein an increase in BRETFect signal in the presence of the agent relative to the absence thereof is indicative that the agent promotes the formation of the ternary or quaternary complex, and wherein a reduction in BRETFect signal in the presence of the agent relative to in the absence thereof is indicative that the agent inhibits the formation of the ternary or quaternary complex.
42. A kit comprising:
one or more vectors for expressing: i. a first protein tagged with a Donor (D) protein, wherein D is a bioluminescent protein (Biol) having an emission spectrum (Biol-Em); ii. a second protein tagged with an Intermediate (I) protein, wherein I is a first Fluorescent protein (FP1) having an excitation spectrum (FP1-Ex) and emission spectrum (FP1-Em); iii. a third protein tagged with an Acceptor (A) protein, wherein A is a second fluorescent protein (FP2) having an excitation spectrum (FP2-Ex) and an emission spectrum (FP2-Em); wherein
a) the FP1-Ex overlaps with the Biol-Em and does not significantly overlaps with the FP2-Ex; b) the FP1-Em overlaps with FP2-Ex; c) the FP2-Ex overlaps with the Biol-Em; and d) the FP2-Em has a longer wavelength than the FP1-Em.
43. A kit comprising:
one or more vectors for expressing: i. a first protein tagged with a first portion of a Donor protein (D1), wherein D1 is a first bioluminescent protein portion (BiolP1); ii. a second protein tagged with a second portion of a Donor protein (D2), wherein D2 is a second bioluminescent protein portion (BiolP2); iii. a third protein tagged with an Intermediate (I) protein, wherein I is a first Fluorescent protein (FP1) having an excitation spectrum (FP1-Ex) and emission spectrum (FP1-Em); iv. a fourth protein tagged with an Acceptor (A) protein, wherein A is a second fluorescent protein (FP2) having an excitation spectrum (FP2-Ex) and an emission spectrum (FP2-Em); wherein said BiolP1 and BiolP2 can form a functional bioluminescent protein (Biol) having an emission spectrum Em if brought in close enough proximity, and wherein
a) the FP1-Ex overlaps with the Biol-Em and does not significantly overlaps with the FP2-Ex; b) the FP1-Em overlaps with FP2-Ex; c) the FP2-Ex overlaps with the Biol-Em; and d) the FP2-Em has a longer wavelength than the FP1-Em.
44. The kit of item 42, wherein said kit comprises one or more vectors comprising a nucleic acid encoding said bioluminescent protein; a nucleic acid encoding said FP1; and a nucleic acid encoding said FP2.
45. The kit of item 43, wherein said kit comprises one or more vectors comprising a nucleic acid encoding said BiolP1; a nucleic acid encoding said BiolP2; a nucleic acid encoding said FP1; and a nucleic acid encoding said FP2.
46. The kit of any one of items 42 to 45, wherein said kit comprises one vector.
47. The kit of item 42 or 44, wherein said kit comprises a first vector for expressing said first protein tagged with Biol, a second vector for expressing said second protein tagged with FP1 and a third vector for expressing said third protein tagged with FP2.
48. The kit of item 43 or 45, wherein said kit comprises a first vector for expressing said first protein tagged with BiolP1, a second vector for expressing said second protein tagged with BiolP2; a third vector for expressing said third protein tagged with FP1 and a third vector for expressing said fourth protein tagged with FP2.
49. The kit of any one of items 42-48, wherein the bioluminescent protein is luciferase, preferably Renilla Luciferase.
50. The kit of any one of items 42-49, wherein the FP1 is mTFP1 or mTagBFP2 fluorescent protein.
51. The kit of any one of items 42-50, wherein the FP2 is Venus, Topaz or mTFP1 fluorescent protein.
52. The kit of any one of item 42-51, further comprising a bioluminescent protein substrate.
53. The kit of item 52, wherein the bioluminescent protein substrate is coelenterazine H or coelenterazine 400a.
54. A cell expressing components (i), (ii) and (iii) defined in any one of items 1-29.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
Unless specifically defined, the terms used in the present application have the meanings that one of ordinary skill in the art would ascribe to them.
In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
BRETFect Biosensors/Assays
Because of the lack of reliable methods for detecting multiprotein complexes (ternary or quaternary) complexes in living cells with sufficient output signal and specificity, the present inventors have developed a new bioluminescence resonance energy transfer (BRET) based system, called BRET-fluorescence enhanced by combined energy transfer (BRETFect), to monitor high order (e.g., ternary, quaternary) complex formation in live cells with high sensitivity and accuracy.
The BRETFect biosensor/method relies on parallel/combined Förster Resonance Energy Transfer mechanisms between at least three molecules: i) a Donor protein (ID or D) i.e., a bioluminescent protein (Biol), such as a luciferase (Luc); ii) an Intermediate protein (IA or I), i.e. a first Fluorescent Protein (FP1) and an Acceptor protein (TA or A), i.e. a second fluorescent protein (FP2).
Thus, the present invention relates to a biosensor for detecting a ternary protein complex comprising:
wherein
The present invention also relates to a method for the detection of a ternary protein complex, the method comprising:
The term “ternary complex” is used herein to refer to a complex comprising 3 or more proteins, which may be the same or different. The biosensor/method described herein may thus be used to detect the formation of a trimer (a complex formed by 3 proteins), or an interaction (direct or indirect) between 3 proteins within a larger protein complex, i.e. comprising 4, 5, 6 or more proteins.
Although the BRETFect assay described herein allows for the highly sensitive detection of ternary complexes, it can further be modified/adapted for quaternary complex detection by combining it with a protein complementation assay (PCA) (see, e.g., Rebois, R. V., et al., Methods, 2008. 45(3): p. 214-8). PCA is a method for the identification of protein—protein interactions in biological systems. In the PCA, the proteins of interest (“Bait” and “Prey”) are each covalently linked to incomplete fragments of a third protein (e.g. DHFR), which acts as a “reporter”. Interaction between the “bait” and the “prey” proteins brings the fragments of the “reporter” protein in close enough proximity to allow them to form a functional reporter protein whose activity can be measured. This principle can be applied to many different “reporter” proteins. Any protein that can be split into two parts and reconstituted non-covalently may be used in a PCA. The two parts are brought together by two interacting proteins fused to them (“bait” and “prey”). Usually enzymes which confer resistance to antibiotics, such as Dihydrofolate reductase or Beta-lactamase, or proteins that give colorimetric or fluorescent signals are used as reporters. When fluorescent proteins are reconstituted, the PCA is called Bimolecular fluorescence complementation assay. The most popular PCAs utilize split versions of the following proteins: Dihydrofolate reductase (DHFR), Beta-lactamase, Yeast Gal4 (as in the classical yeast two-hybrid system), Luciferase, Split TEV (Tobacco etch virus protease), Ubiquitin, GFP (split-GFP), LacZ (beta-galactosidase). In an embodiment, the reporter protein is a luciferase and the biosensor comprises a split version of the luciferase, i.e. two fragments/portions of the luciferase that can generate a functional luciferase when the two fragments/portions are brought in close enough proximity. Fragments of luciferase suitable for PCA are disclosed, for example, in Luker and Luker, Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice, Molecular Imaging, Methods in Molecular Biology 680, Chapter 2, and include those depicted in the Table II below.
Renilla luciferase
Renilla luciferase
Gaussia luciferase
The BRETFect biosensor/method of the present invention may thus be conveniently adapted to detect quaternary complexes by including a PCA element. For example, one part of the luciferase enzyme could be coupled to a first interacting partner (ID) and the second part to a 2nd interacting partner. FP1 and FP2 would be coupled to 3rd and 4th interacting partners. Upon interaction between the first and 2nd interacting partners, a functional luciferase would be recreated, thereby enabling the transfer to the intermediate and terminal acceptors. Certain nuclear receptors are known to form multimers of higher order than dimers. To monitor activity of higher order complexes, nuclear receptor 1 (NR1) and NR2 can be tagged with half luciferases, NR3 with FP1 (e.g., mTFP1) and a cofactor with FP2 (e.g., eYFP).
Thus, the present invention further relates to a biosensor for the detection of a quaternary protein complex comprising the elements defined above, wherein one of Biol, FP1 or FP2 is divided in two parts/portions that can generate a functional protein when brought in close enough proximity, and the biosensor comprises an additional tagged protein.
Accordingly, in another aspect, the present invention relates to a biosensor for the detection of a quaternary protein complex comprising:
In another aspect, the present invention relates to a method for the detection of a quaternary protein complex, the method comprising:
In an embodiment, FP1-Ex and FP2-Ex are such that FP1 and FP2 are excited by a different portion of the Biol-Em spectrum, i.e. the transfers from D to I and A are complementary rather than competitive. In an embodiment, FP1-Ex overlaps with the lower end (i.e. lower wavelengths) of the Biol-Em spectrum and FP2-Ex overlaps with the higher end (i.e. higher wavelengths) of the Biol-Em spectrum.
The term “quaternary complex” is used herein to refer to a complex comprising 4 or more proteins, which may be the same or different. The biosensor/method described herein may thus be used to detect the formation of a complex formed by 4 proteins, or an interaction (direct or indirect) between 4 proteins within a larger protein complex, i.e. comprising 5, 6, 7 or more proteins.
As used herein, the terms first, second and third proteins are meant to refer to the three interacting components in a ternary complex that is to be detected in accordance with the biosensor/method described. As used herein, the terms first, second, third and fourth proteins are meant to refer to the four interacting components in a quaternary complex that is to be detected in accordance with the biosensor/method described.
As used herein, the terms “first Donor” (D1) and “second Donor” (D2) are meant to refer to 2 complementary parts of the donor protein that is suitable to generate/reconstitute a functional or active donor protein when brought in close proximity by direct or indirect interaction between the proteins to which they are bound. D more specifically refers to a bioluminescent enzyme, for example a luciferase. The term “Intermediate” (I) refers to a first fluorescent protein (FP1), which following excitation by the D, transfers part of its energy to the Acceptor (A). The “Acceptor” (A) is a fluorescent protein (FP2) that is different from FP1 (and which may be excited by the energy emitted by both D and I).
Thus, the first, second and third proteins (or first, second, third and fourth proteins) may be the same protein/peptide (to detect a homotrimer or “homoquatermer” of a protein, which may or may not be in a larger protein complex), 3 or 4 different proteins (to detect a heterotrimer or “heteroquatermer”, which may or may not be in a larger protein complex), or combinations thereof (e.g., to detect a complex comprising 2 or 3 identical proteins and a 3rd (or 3rd and 4th) distinct protein(s)). In an embodiment, the first, second and third proteins (or first, second, third and fourth proteins) are identical. In another embodiment, the first, second and third proteins (or first, second, third and fourth proteins) are all different. In another embodiment, the first and second proteins are identical and the third protein is different. In another embodiment, the first and third proteins are identical and the second protein is different. In another embodiment, the second and third proteins are identical and the first protein is different. It should be understood that for a given ternary complex, the proteins fused to the bioluminescent enzyme, first fluorescent protein and second fluorescent protein (D, I and A, respectively) may be interchanged. For example, for detecting the formation of a ternary complex comprising protein1, protein2 and protein3, several configurations are possible: (1) protein1 is tagged with D, protein2 is tagged with I and protein3 is tagged with A; (2) protein1 is tagged with D, protein2 is tagged with A and protein3 is tagged with I; (3) protein1 is tagged with I, protein2 is tagged with D and protein3 is tagged with A; (4) protein1 is tagged with I, protein2 is tagged with A and protein3 is tagged with D; (5) protein1 is tagged with A, protein2 is tagged with I and protein3 is tagged with D; or (6) protein1 is tagged with A, protein2 is tagged with D and protein3 is tagged with I.
The biosensor/method is based on the simultaneous energy transfer between a bioluminescent protein (e.g., luciferase) donor and intermediate and terminal acceptors, appropriately chosen to also enable transfer from the intermediate to terminal acceptor while minimizing contaminating signals. This specific energy transfer mechanism was shown to greatly improve sensitivity of detection.
As used herein, the term “bioluminescent protein” refers to a class of enzymes that are able to catalyze (e.g., oxidize) a substrate, which in turn emits fluorescent light upon catalysis (oxidation). Examples of bioluminescent proteins include lumazin, obelin, aequorin and luciferase (native and functional variants thereof). In an embodiment, the bioluminescent protein is luciferase.
As used herein, the term “luciferase” refers to the class of oxidative enzymes used in bioluminescence and which is distinct from a photoprotein. One example is the firefly luciferase (EC 1.13.12.7) from the firefly Photinus pyralis (P. pyralis luciferase). Several recombinant luciferases from several other species including luciferase from Renilla reniformis (GENBANK: AAA29804) and variants thereof (e.g., a stable variant of Renilla Luciferase e.g., RlucII (GENBANK: AAV52877.1), Rluc8 (GENBANK: EF446136.1) and Gaussia Luciferase (Gluc, GENBANK: AAG54095.1) are also commercially available. Any luciferase can be used in accordance with the present invention as long as it can metabolize a luciferase substrate such as luciferins. Luciferins are a class of light-emitting heterocyclic compounds that are oxidized in the presence of luciferase to produce oxyluciferin and energy in the form of light. Non-limiting examples of luciferins include D-luciferin, imidazopyrazinone-based compounds such as coelenterazine (coelenterazine 400a (DeepBlueC™) and coelenterazine H), ViviRen™ (from Promega®), Latia luciferin ((E)-2-methyl-4-(2,6,6-trimethyl-1-cyclohex-1-yl)-1-buten-1-ol formate), bacterial luciferin, Dinoflagellate luciferin, etc. Luciferase substrates may have slightly different emission spectra and will thus be selected to favor the optimal energy transfer to the initial and terminal acceptors. In an embodiment, the luciferase is wild-type (or native) Renilla Lucificerase. In an embodiment, the luciferase is a variant of Renilla luciferase, e.g., Rluc8 or RLucII. In a specific embodiment the luciferase is RLucII and the luciferin is coelenterazine H.
As used herein, the term “fluorescent protein” refers to any protein which becomes fluorescent upon excitation at an appropriate wavelength. A broad range of fluorescent proteins have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Non-limiting examples of green Fluorescent Protein include EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen and T-Sapphire. Non-limiting Examples of blue fluorescent protein include EBFP, EBFP2, Azurite and mTagBFP. Non-limiting examples of Cyan Fluorescent proteins include ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal). Non-limiting examples of Yellow fluorescent proteins include EYFP, Topaz, Venus, mVenus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1 and mBanana. Non-limiting Examples of orange fluorescent proteins include Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer and mTangerine. Non-limiting Examples of red fluorescent porteins include mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum and AQ143.
The bioluminescent protein and fluorescent proteins are covalently attached to the first, second and third proteins. In an embodiment, the bioluminescent protein and fluorescent proteins forms a fusion protein with the first, second and third proteins. The bioluminescent protein and/or fluorescent proteins may be fused N-terminal, within, or C-terminal relative to first, second and/or third proteins. In embodiments, the bioluminescent protein and/or fluorescent proteins may be covalently linked to the first, second and/or third proteins either directly (e.g., through a peptide bond) or “indirectly” via a suitable linker moiety, e.g., a linker of one or more amino acids (e.g., a polyglycine linker) or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc. In an embodiment, one or more additional domain(s) may be inserted before (N-terminal), between or after (C-terminal) the bioluminescent-fluorescent proteins and the first, second and/or third proteins.
Any combination of bioluminescent protein (e.g., luciferase) and fluorescent proteins may be used in accordance with the present invention as long as the requirements/criteria defined herein are met. In embodiments, the excitation spectrum of I (FP1-Ex) overlaps part of the Luc (D) emission spectrum (Luc-Em) and overlaps minimally with FP2 excitation spectrum (FP2-Ex). Also, the emission spectrum of I (FP1-Em) overlaps part of the A excitation spectrum (FP2-ex), and optimally its peak should be closer to that of the Luc-Em spectrum than to that of the FP2-em. As for the A, its excitation spectrum overlaps part of the D (Luc) emission spectrum and its emission spectrum is red-shifted (longer wavelength) from the I (FP1) emission spectrum, allowing for selective monitoring of FP2 emission.
Several bioluminescent and fluorescent proteins and their excitation and emission spectra are known in the art and can thus be used to identify a suitable combination of bioluminescent protein (e.g., luciferase), FP1 and FP2 (see Table I below). The Fluorescent proteins selected (FP1 and FP2) should not substantially interact to form homodimers or heterodimers or multimers under the experimental conditions used for the BRETFect assay. One representative suitable combination is Renilla Luciferase as the Donor (D), mTPF1 fluorescent protein as the Intermediate (I) and Venus fluorescent protein as the Acceptor (A). Another suitable combination is Renilla Luciferase (RLucII-coel400) as the Luciferase (D), mtagBFP2 (PDB) as the I and mTFP1 as the A (with measures at 400 and 530 nm). As luciferase substrates may have slightly different emission spectrum, the luciferase substrate is preferably selected to optimize energy transfer between the luciferase and intermediate and terminal acceptors.
As used herein, the term “BRETFect signal” (or “BRETFest signal”) is meant to refer to the specific BRET signal which is observed between two proteins in the presence of a third interacting partner in accordance with the present invention. The BRETFect signal is measured as the difference between the ratios of the Acceptor (A) emission over Initial Donor (D) emission in a condition containing D, I and A and the sum of the ratios measured with D-I and D-A alone. A positive BRETFect signal is indicative of the formation of a ternary or quaternary complex.
“Overlap” as used in the context of the present invention refers to the ability of the emitted light from a donor luminescent enzyme (e.g., luciferase) to be of a wavelength capable of excitation of a fluorophore placed in close proximity and/or to the ability of the emitted light from a fluorophore to be of a wavelength capable of excitation of another fluorophore placed in close proximity (usually within 100 Å). In an embodiment, the overlap means that the overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor (i.e. the % of the area of the excitation spectrum of the acceptor that is “shared with” the emission spectrum of the donor, as illustrated by the black arrow shared by Luc-Em and FP1-Ex in
The term “overlap minimally” or “does not significantly overlap” means that the overlap between the excitation spectrum of the donor (FP1) and the excitation spectrum of the acceptor (FP2) is 35% or less, preferably 30, 20, 15%, 10%, or 5% or less. In an embodiment, “overlap minimally” or “does not significantly overlap” means that the overlap is 30% or less. In an embodiment, “overlap minimally” or “does not significantly overlap” means that the overlap is 25% or less. In an embodiment, “overlap minimally” or “does not significantly overlap” means that the overlap is 20% or less. In an embodiment, “overlap minimally” or “does not significantly overlap” means that the overlap is 15% or less. In an embodiment, “overlap minimally” or “does not significantly overlap” means that the overlap is 10% or less.
In another embodiment, “overlap minimally” or “does not significantly overlap” means that the excitation spectrum of the donor (FP1-Ex) does not overlap with the maximal or peak excitation wavelength of the acceptor (FP2-Ex). The mTFP1-Venus is a representative combination of fluorophores that fulfill this condition of “minimal overlap”. The excitation spectrum of mTFP1 (FP1) ranges from about 400 nm to about 500 nm (maximum=about 462 nm), and the maximal excitation wavelength of Venus (FP2) is about 515 nm, thus the upper limit of the excitation spectrum of mTFP1 (500 nm) does not overlap with the maximal excitation wavelength of Venus (515 nm).
The BRETFect biosensor/method can measure the combined interaction between the D, I and A together or each pair separately provided three different conditions are present:
i. Presence of D and A. In this condition D and A interaction can be monitored by BRET using appropriate filter combinations;
ii. Presence of D and I. In this condition D and I interaction can be monitored by BRET using appropriate filter combinations (different from above);
iii. Presence of D, I and A. In this condition I-A interactions can be monitored by Fluorescence Resonance Energy Transfer using standard methods. Moreover D, I and A interaction can be monitored by BRETFect as the BRET signal between D and A is amplified by the presence of I in the complex.
As shown in
To measure the BRETFect signal, the ratio of A emission over D emission is measured with filter sets chosen according to the specificity of the Biol (e.g., Luc) and FP1/2 used in the assay. The A/FP2 emission filter can be centered on its emission peak to increase signal but a filter that is more red-shifted to decrease contamination from FP1 emission allows for better measurements. The D-Biol/Luc emission filter can be chosen to avoid the part of the Biol- (e.g., Luc) Em spectrum that is absorbed by the Intermediate (I) without compensating emission in the same detection channel, in order to avoid artifactual signals due to quenching of the Biol (e.g., Luc) emission by FP1 upon interaction of D and I. In an embodiment, the Biol- (e.g., Luc-) Em has a longer wavelength than the FP1-Ex. In an embodiment, FP1-Ex does not overlap with FP2-Ex. In an embodiment, this is no or significantly no overlap between FP2-Em and FP1-Em.
The BRETFect signal is defined as the difference between the ratio of A over D emission in the ternary/quaternary condition and the sum of similar measurements for contaminating signal from D-I and D-A performed using the same A/FP2 and D/Biol- (e.g., Luc) Em filters. A positive BRETFect signal indicates formation of a ternary or quaternary complex.
In an embodiment, D is RLucII, I is mTFP1 and A is Venus, and the luciferase substrate is Coelenterazine H. In another embodiment, D is RLucII, I is mtagBFP2 (PDB) and A is mTFP1, and the luciferase substrate is Coelenterazine 400a.
BRETFect combines energy transfer between a luciferase Donor and an Intermediate and an Acceptor, appropriately chosen to also enable transfer from the Intermediate to the Acceptor while minimizing contaminating signals. This specific energy transfer mechanism was shown to greatly improve signal detection. Further, titration experiments can provide information on the stoechiometry of the complex. The biosensor/method described herein is broadly applicable to the detection of any complex involving at least three proteins (same or different) such as those formed by nuclear receptors (and/or nuclear receptor-interacting proteins), GPCRs (and/or GPCR-interacting proteins) and Tyrosine Kinase receptors (RTKs) (and/or RTK-interacting proteins). There are several examples of protein complexes involving three or more protein/peptides molecules in various signalling pathways, such as those illustrated in
In an embodiment, the biosensor/method described herein may be used to detect ternary or quaternary complexes involving one or more nuclear receptors. Accordingly, in an embodiment, at least one of the first, second, third or fourth protein is a nuclear receptor protein or a suitable fragment thereof, i.e. a fragment that maintains the ability to oligomerize (e.g., dimerize, trimerize) and/or to interact with one or more of its binding partners to form a ternary or quaternary complex.
As used herein, the term “nuclear receptor” refers to a class of proteins found within cells that are responsible for sensing steroid and thyroid hormones and certain other molecules. In response, these receptors work with other proteins to regulate the expression of specific genes, thereby controlling the development, homeostasis, and metabolism of the organism. Non-limiting examples of nuclear receptors include: Estrogen receptor (ERα and ERβ), Retinoic acid receptors (RXRγ, RARα), orphan receptor Nurr77, Androgen receptor (AR), Glucocorticoid receptor (GR), Progesterone receptor (PR), etc. Nuclear receptors are involved in the formation of ternary complexes involving receptor dimers and and coactivator/corepressor proteins (6-10), which can be detected using the biosensor/assay described herein. An embodiment uses estrogen receptors alpha (ERα) or beta (ERβ) as homo- or heterodimers. Another embodiment uses RAR and RXR.
Nuclear receptors have the ability to directly bind to DNA and regulate the expression of adjacent genes, hence these receptors are classified as transcription factors. The regulation of gene expression by nuclear receptors generally only happens when a ligand—a molecule that affects the receptor's behavior—is present. More specifically, ligand binding to a nuclear receptor results in a conformational change in the receptor, which, in turn activates the receptor, resulting in up-regulation or down-regulation of gene expression.
In an embodiment, at least two, or two, of the first, second third and/or fourth proteins are nuclear receptor proteins or suitable fragments thereof. The at least two, or two nuclear receptor proteins or fragments may be the same nuclear receptor proteins or fragments (e.g., ERα-ERα; ERβ-ERβ) or different nuclear receptor proteins or fragments or different (e.g., ERα-ERβ).
Nuclear receptors bound to hormone response elements recruit a significant number of other proteins (referred to as transcription coregulators) that facilitate or inhibit the transcription of the associated target gene into mRNA. The function of these coregulators (coactivators, corepressors), are varied and include chromatin remodeling (making the target gene either more or less accessible to transcription) or a bridging function to stabilize the binding of other coregulatory proteins. Nuclear receptors may bind specifically to a number of coregulator proteins, and thereby influence cellular mechanisms of signal transduction both directly, as well as indirectly. Non-limiting examples of coregulatory proteins or derived peptides/domains include the p160 steroid receptor coactivators, the p300/CBP coactivators, the NCoR or SMRT corepressors. In addition, protein modifiers such as ubiquitin and SUMO can also be used in this system to monitor covalent modifications of nuclear receptors. In an embodiment, at least one of the first, second third or fourth protein is a nuclear receptor interacting domain or nuclear receptor interacting domain (NID)-containing protein, such as a nuclear receptor coactivator protein or a nuclear receptor interacting fragment thereof. Nuclear receptor interacting domains typically contain one or several LXXLL motifs. Examples of NID-containing protein include steroid receptor coactivator (SRC) proteins such as nuclear receptor coactivator 1 (NCOA1 or SRC1), nuclear receptor coactivator 2 (NCOA2 or SRC2) and nuclear receptor coactivator 3 (NCOA3 or SRC3), activating signal cointegrator-2 (ASC-2), TRAP220, TIP60, TIF1α and PGC1. In an embodiment, the NID-containing protein comprises the NID of NCOA1 or NCOA2. In further embodiment, the NID or NID-containing protein comprises the amino acid sequence of SEQ ID NO:10 or 11.
In an embodiment, the biosensor/method described herein may be used to detect ternary or quaternary complexes involving one or more GPCRs and/or G protein subunits (i.e. one or more of the first, second third and/or fourth proteins is/are GPCRS and/or G protein subunits). The term “GPCR” refers to a class of integral membrane proteins that possess seven membrane-spanning domains or transmembrane helices and which signal through G proteins. As used herein, the term “GPCR” encompasses full length native GPCR molecules as well as mutant GPCR molecules (e.g., fragments or variants of native GPCRs) that maintain the ability to dimerize/oligomerize and/or interacts with binding partners in a manner that can be modulated by ligands (11). In an embodiment, the GPCR is a native GPCR. Examples of GPCRs include 5-Hydroxytryptamine receptors, Acetylcholine receptors (muscarinic), Adenosine receptors, Adhesion Class GPCRs, Adrenoceptors, Angiotensin receptors, Apelin receptor, Bile acid receptor, Bombesin receptors, Bradykinin receptors, Calcitonin, receptors, Calcium-sensing receptors, Cannabinoid receptors, Chemerin receptor, Chemokine receptors, Cholecystokinin receptors, Class Frizzled GPCRs, Complement peptide receptors, Corticotropin-releasing factor receptors, Dopamine receptors, Endothelin receptors, Estrogen (G protein-coupled) receptor, Formylpeptide receptors, Free fatty acid receptors, GABAB receptors, Galanin receptors, Ghrelin receptor, Glucagon receptor family, Glycoprotein hormone receptors, Gonadotrophin-releasing hormone receptors, Histamine receptors, Hydroxycarboxylic acid receptors, Kisspeptin receptor, Leukotriene receptors, Lysophospholipid (LPA) receptors, Lysophospholipid (S1P) receptors, Melanin-concentrating hormone receptors, Melanocortin receptors, Melatonin receptors, Metabotropic glutamate receptors, Motilin receptor, Neuromedin U receptors, Neuropeptide FF/neuropeptide AF receptors, Neuropeptide S receptor, Neuropeptide W/neuropeptide B receptors, Neuropeptide Y receptors, Neurotensin receptors, Opioid receptors, Orexin receptors, Oxoglutarate receptor, P2Y receptors, Parathyroid hormone receptors, Peptide P518 receptor, Platelet-activating factor receptor, Prokineticin receptors, Prolactin-releasing peptide receptor, Prostanoid receptors, Proteinase-activated receptors, Relaxin family peptide receptors, Somatostatin receptors, Succinate receptor, Tachykinin receptors, Thyrotropin-releasing hormone receptors, Trace amine receptor, Urotensin receptor, Vasopressin and oxytocin receptors, VIP and PACAP receptors. A list of GPCRs is given in Foord et al. (12) and an updated list of GPCRs is available in the IUPHAR-DB database (13,14).
GPCRs and G protein subunits (e.g., Gβ, Gα and Gγ) are known to interact directly or indirectly and form multiprotein complexes with several accessory and signaling proteins such as G-protein-coupled receptor kinases, other cell surface receptors or membrane proteins, βarrestin, regulators of G-protein signalling (RGS) proteins (RGS1 to 22), activators of G-protein signalling (AGS) proteins (also referred to as G-protein-signaling modulator (GPSM) proteins, resistance to inhibitors of cholinesterase 8 proteins (Ric-8), etc. Examples of ternary complexes involving one or more GPCRs and/or G protein subunits that may be detected using the biosensor/method described herein include GPCR-Gα-Gγ, GPCR-GRK-Gα, GPCR-GRK-Gβ, GPCR homodimer-Gα, GPCR heterodimer-Gα, GPCR homodimer-Gβ, GPCR heterodimer-Gβ, GPCR homodimer-βarrestin, GPCR heterodimer-βarrestin, GPCR-Gβ-βarrestin, GPCR-Gα-Gβ, GPCR-GRK-Gβ, GPCR-membrane protein-Gα, GPCR-membrane protein-Gβ, GPCR-membrane protein-Gγ, GPCR-membrane protein-GRK, GPCR-membrane protein-βarrestin, GPCR-membrane protein-RGS, GPCR-RGS-Gα and GPCR-AGS-Gα. Examples of membrane proteins that interact directly or indirectly (e.g., through scaffold proteins such as RGS proteins) with GPCRs include RAMP1, RAMP2, RAMP3, LRP5, LRP6, certain RTKs (FGFR1, EGFR) and adenylate cyclase.
Screening Assays
The biosensor/method described herein can advantageously be used to identify compounds which modulate the formation of ternary or quaternary complexes. Formation of ternary or quaternary complexes can be tested by BRETFect in the presence and absence of one or more test compounds. An increase or a decrease in the BRETFect signal in the presence of a test compound indicates that the compound is able to modulate (i.e., increase or decrease, stabilize or inhibit) the formation of the ternary or quaternary complex. Potencies of different ligands for ternary or quaternary complex assembly can be measured, and when different isoforms of the components of ternary or quaternary complexes are possible, allosteric effects of each monomer isoform on ligand-dependent trimer formation can be detected by comparing ligand potencies with ternary or quaternary complexes containing different isoforms.
Thus, in another aspect, the present invention relates to a method for determining whether an agent (e.g., a molecule, compound or ligand) modulates the formation of a ternary or quaternary protein complex comprising performing the method defined above in the presence and in the absence of said agent, wherein an increase in BRETFect signal in the presence of the agent relative to the absence thereof is indicative that the agent promotes the formation of the ternary or quaternary protein complex, and wherein a reduction in BRETFect signal in the presence of the agent relative to in the absence thereof is indicative that the agent inhibits the formation of the ternary or quaternary protein complex.
As used herein, the terms “molecule”, “compound”, “agent” or “ligand” are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term “molecule” therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like. Non-limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents. The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling. A test compound is a compound which is tested to determine whether it can modulate (increase or decrease, stabilize or inhibit) the formation of ternary or quaternary complexes in accordance with the present invention.
In accordance with the present invention, any test compound suspected of modulating the activity/formation of a ternary or quaternary complex can be used. The compounds can be tested individually or several test compounds may be tested at the same time.
The method of the present invention can also be used to identify small interfering RNAs (shRNAs, siRNAs) or cDNAs which modulate the formation of ternary or quaternary complexes.
BRETFect assays were shown to be sufficiently robust to be amenable to high-throughput screening assays of small molecule libraries, small interfering RNA libraries or cDNA libraries (see, e.g.,
Kits
The present invention also encompasses kits which can be used to monitor the formation of high order (e.g., ternary complexes) in cells using BRETFect. Kits of the present invention may comprise:
In an embodiment, the kit comprises:
The kit may also be adapted for detecting higher order complexes (e.g., quaternary complexes) by adapting the kit to include a PCA component, as described herein. In such an embodiment, one of the reporter proteins (e.g., FP1) would be divided in two parts, which when placed into close proximity can reconstitute a functional protein non-covalently. Such a kit could thus comprise for example one or more vectors for expressing two proteins each tagged with luciferase portion (Luc). Other reporter proteins may also be used in accordance with the present invention.
The vector(s) above permit the cloning of nucleic acids encoding proteins of interest (which form or are suspected to form a complex) so as to allow expression of these proteins of interest tagged with a bioluminescent protein (e.g., luciferase), FP1 and FP2. The formation of a complex between the proteins of interest may be monitored using the biosensor/method described herein.
Optionally, the kit will further comprise suitable luciferase substrate and instructions for detecting a ternary or quaternary complex by BRETFect. The kit may also comprise any other reagent suitable for the purpose of the kit. For example, transfection reagents may also be included. Also, the kit may further comprise ligands which are normally necessary to promote the activity and formation of a ternary or quaternary complex. For example nuclear receptors ligands (estrogen, retinoic acid, lipids, testosterone, progesterone, etc.) or GPCR ligands may further be included in the kit.
Cells Expressing Fusion Proteins Comprising D (D1, D2), I and A
The present invention also provides cells expressing a suitable combination of proteins tagged with Donor (D) (or D1 and D2), Intermediate (I) and Acceptor (A) proteins to monitor formation of a ternary or quaternary complex using the biosensor/method described herein. As indicated above, when a PCA component is incorporated in the biosensors, methods and kits disclosed herein (e.g., to detect a quaternary complex), the cells will express at least one of D, I and A in portions (e.g., two portions) which when expressed may reconstitute a functional protein non-covalently when placed into close proximity (upon interaction/oligomerization of the two portions). The cells will express D, I and A and control cells expressing either (i) D and I, (ii) I and A and (iii) D and A may also be provided in order to monitor BRETFect signal and formation of ternary complexes. The specific type of cells used will be chosen in accordance with the specific ternary complex and associated activity that is detected.
In the following non-limiting Examples, effectiveness of the BRETFect assay in accordance with embodiments of the present invention is illustrated by demonstrating that ligands specific for estrogen receptor alpha (ERα) can activate α/β heterodimers and that each monomer allosterically regulates the other. BRETFect was validated for the robust, HTS-compatible detection of coactivator recruitment and SUMO modification on estrogen receptor homo- and heterodimers in live cells. BRETFect notably revealed recruitment of AF1-interacting to activation of ERα/β heterodimers by the ERα-specific ligand PPT and an allosteric control of coactivator recruitment between liganded dimeric partners in live cells.
Cell Lines, Plasmids and Reagents
HEK293 cells (Sigma-Aldrich®, Oakville, Ontario, Canada) were grown in DMEM with 10% fetal bovine serum (Wisent® Inc., St-Bruno, Quebec, Canada). Polyethyleneimine (25 kDa molecular mass, linear or branched forms) was obtained from Sigma-Aldrich®. Coelenterazine H and Coelenterazine 400a were obtained from NanoLight Technology® (Pinetop, Ariz.). 17-beta-Estradiol (E2) and 4-hydroxytamoxifen (OHT) were purchased from Sigma-Aldrich®, RU58668 (RU58), ICI182,780 (ICI182) and raloxifene (Ral) were purchased from Tocris® Cookson Ltd (Minneapolis, Minn.). pcDNA-RLucII vectors are described in (4). pCMV-Venus and pCMV-mTFP1 were generated by amplification of the relevant fluorophore genes and replacement of the eGFP in the peGFP-N1 vector (PerkinElmer Corp., Wellesley, Mass.). ERα/β and Nur77 cDNAs were cloned into the above-described vectors by PCR amplification of coding sequence and digestion of 5′ and 3′ ends with appropriate restriction enzymes. The coactivator construct was generated by inserting oligonucleotides coding for a repeat of the first NCOA2 LXXLL motif (WT sequence: GAT CTA ACC ATG AAG CAT AAA ATT TTG CAC AGA CTC TTG CAG GAC AGC AGT CTC GAG ATG AAG CAT AAA ATT TTG CAC AGA CTC TTG CAG GAC AGC AGT CTC GAG, SEQ D NO: 1; non-interacting mutant sequence: GAT CTA ACC ATG AAG CAT AAA ATT GCG CAC AGA GCC GCG CAG GAC AGC AGT CTC GAG ATG AAG CAT AAA ATT GCG CAC AGA GCC GCG CAG GAC AGC AGT CTC GAG, SEQ D NO: 2) and a NLS sequence derived from GR (sequence: GAT CGA GCC CAC TCC ACA CCT CCA AAA AAC AAA CGA MC GTT CGA GAT CCC AAG GAT CGA GCC CAC TCC ACA CCT CCA AAA AAC AAA CGA AAC GTT CGA GAT CCC MG, SEQ D NO: 3) into pCMV-Venus. The ERα(L507R) mutant was created by overlapping primer site-directed mutagenesis. The SRC1-RID was cloned from NCOA1 cDNA with primers flanking the third and fifth LXXLL motif in 5′ and 3′ respectively (positions 2155 to 2517 in NCBI Reference Sequence NM_003743—oligo sequence: ATG TAC TCT CAA ACC AGT CAC AAA, SEQ D NO: 4) and TGA GGG GCT ACC CTC CTG, SEQ D NO:5). The AF1-interacting domain (AF1ID) encompasses the glutamine rich region of SRC1, cloned from NCOA1 cDNA positions 3129 to 3782 into Venus expression vector as for CoApep. SUMO3-Venus was obtained from Dr M. K. Chelbi-Alix, French National Centre for Scientific Research, France.
HEK293 Cell Transfection
HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in 175 cm2 culture flasks. Two days before experiments, HEK293 cells were harvested and switched to phenol red-free DMEM containing 10% charcoal-stripped serum. HEK293 cells were transfected via PEI with 1.66 μg of DNA, 1.66 μg PEI-branched and 5 μg PEI-linear per 106 cells. DNA mixes and PEI dilutions were made in a total volume of 75 ml PBS separately before mixing. After 10 minutes incubation, HEK293 cell suspensions (1.25×106/ml) were added directly to the DNA-PEI transfection mixes (850 μl of cells per 150 μl of transfection mix) and 100 μl for DNA-PEI-cell suspensions was aliquoted per well in 96-well white-bottom culture plate (Corning) and grown for 48 hours.
Bioluminescence Resonance Energy Transfer Assays
Unless otherwise indicated, BRET assay transfection mixes contained 150 ng of RLuc-tagged donor and 0 to 1.5 μg of YFP-tagged acceptor for titrations or 1.5 μg for single point experiments. 48 hours after transfection cells were washed with PBS and treated with specified ligands or vehicle (0.1% DMSO) in 100 μl PBS for 40 minutes at 37° C. Coelenterazine H was added to a final concentration of 5 μM, and readings were immediately collected on a Mithras™ LB 940 (Berthold Technologies™, Bad Wildbad, Germany), with sequential integration of signals detected at 485 nm (Renilla luciferase emission) and LP550 nm (YFP emission). BRET ratios displayed for titration curves are the stimulated YFP emission (exc 485 nm, em LP550 nm) acquired with a FlexStation™ 3 Microplate Reader (Molecular Devices®) divided by Luciferase emission as recorded by the Mithras™ LB 940 in the 485 nm channel. Net BRET values (BRET ratios for fused proteins minus BRET ratios with fused Luciferase but unfused GFP) are represented as a function of Log10([Fluorescent protein]/Luc). Emission spectrum experiments (
SRET, BRETFect and FRET Assays
Unless otherwise indicated, BRETFect and SRET assay transfection mixes contained 100 ng of LucII tagged donor, with or without 400 ng of mTFP1 tagged I and/or 1 μg of Venus tagged acceptor (total DNA concentration kept constant with pcDNA3.1-Hygro). For spectral analysis of emission (
An aim of the present studies was to develop BRET assays that reliably monitor ternary complexes without the need for spectral unmixing and with robust signal output to enable the dissection of ternary protein complexes and application to high-throughput screens. One of the limitations of SRET is the need to transfer energy sequentially from the D (RLucII activated with Coelenterazine 400a for SRET) to the I (uvGFP) and subsequently from the I to the A (eYFP) (see
The use of coelenterazine H changes the pattern of energy transfer: while SRET relies on sequential transfer of energy from the RLucII to the I (uvGFP) and subsequently to the A (eYFP) (Carriba, P., et al., supra), here energy from the RLucII is transferred in a combined manner to I (mTFP1) and A (Venus). Importantly, the energy emitted from RLucII in the shorter wavelengths of the spectrum (400 to 460 nm), which is not favorable for A excitation, can be efficiently transferred to the I mTFP1, which will emit at 492 nm, a more favorable emission for Venus. Hence, Bioluminescence Resonance Energy Transfer with Fluorescence Enhancement by Combined Energy Transfer (BRETFect) monitors an amplification of the BRET increase in a donor+I+A condition versus the donor+A control condition.
To fully characterize the pharmacological properties of ER ligands, assays that could reliably detect AF1 and AF2 coactivator binding were perfected (
Fluorescence Resonance Energy Transfer (FRET) and BRET both detect protein-protein interactions in real-time in intact cells (21,22). FRET allows reliable monitoring of sequential transfer between three fluorophores in a three-color or triple-FRET assay (23,24), but suffers from problems due to photobleaching and contaminating cross-excitation requiring spectral unmixing, which complicates high-throughput screening applications. Our aim was to develop a BRET assay that reliably monitors ternary complexes with robust signal output and without need for spectral unmixing. BRETFect achieves this by using a Donor (D)-Intermediate (I)-Acceptor (A) setup where the Acceptor receives energy directly from the Donor and indirectly via the Intermediate in a combined transfer. The Intermediate is chosen such that it can act as a relay to increase total energy transfer from the Donor to the Acceptor, by shifting parts of the emission spectrum that are poorly used by the Acceptor to wavelengths that are more readily absorbed. For instance, using mTFP1 as an Intermediate should enable efficient transfer of the energy emitted by the donor RLucII in the shorter wavelengths of its spectrum (400 to 460 nm), to wavelengths peaking around 492 nm, more favorable for Venus excitation (
Negative controls for the contribution of the Intermediate in energy transfer should include the intermediate interaction partner in the absence of fusion with the fluorophore and mutant proteins that cannot interact with the donor or with the acceptor. In addition, choosing the fusion partner protein of the Intermediate as a more efficient interactor with the Acceptor compared to the Donor will also result in a more important contribution to the overall signal.
Characterization or development of dimer-selective ligands is an important pharmacological priority in the development of novel therapeutics, but necessitates assays that can monitor activity of specific homodimers and heterodimers. Applicants thus determined whether BRETFect assays can distinguish between ERα/β homo- and hetero-dimers.
As a proof of concept, the agonist-dependent recruitment of AF2-binding CoApep tagged with Venus by homo- and hetero-dimers of nuclear receptors ERα and ERβ, tagged with RLucII and mTFP1 (10,25,26) was monitored (
BRET ratios for the binary and ternary complexes in the presence of E2 or of the antiestrogen 4-hydroxytamoxifen (OHT) were monitored, the latter being permissive for ER dimer formation but repressing coactivator recruitment (27,28). In the control ERα-RLucII+CoApep-Venus condition, treatment with E2 produced a robust BRET signal, reflecting complex formation, while OHT decreased basal CoApep recruitment (
Both ERα and ERβ recruited CoApep-Venus in an E2-induced, OHT-repressed manner in BRET assays (
The impact of SERDs on ERβ SUMOylation was not previously addressed. In BRET assays, SERDs did not induce BRET signals between SUMO3-Venus and ERβ, contrary to ERα (
Using BRETFect assays for recruitment of CoApep-Venus by different combinations of ERα and ERβ fused to RLucII or mTFP1 (see above), it was tested whether the ERα-specific agonist propylpyrazole triol (PPT) or the ERβ-selective agonist diarylpropionitrile (DPN) (29-31) can lead to heterodimer activation. PPT activated ERα, but not ERβhomodimers (
Serial resonance energy transfer (SRET) assays have been previously developed to address the detection of trimetric complexes. Similar to BRETFect, this approach involves a luciferase as donor and fluorescent proteins as Intermediate (uvGFP) and Acceptor (eYFP), but this approach posits an absence of direct transfer from D to A (see
As screening methods are becoming readily available and encompass increasing numbers of natural and synthetic molecules, the possibility of targeting a specific trimeric complex, such as a nuclear receptor heterodimer in complex with a coactivator, in real-time in live cells should prove a tremendous asset. Discovery of dimer-selective nuclear receptor ligands is an important pharmacological opportunity (33-36) that necessitates development of robust assays for the activity of specific homo- and heterodimers. As shown in
The present inventors were also able to detect the formation of a ternary complex between RXRγ, RARα, and a corepressor (CoR) by using the BRETFect assay as described above. Results are presented in
Thus, inventors have demonstrated herein that BRETFect is a powerful tool to monitor formation of high order (e.g., ternary) complexes.
The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
The present application is a National Entry Application of PCT application no. PCT/CA2014/051266 filed on Dec. 23, 2014, which itself claims the benefit of U.S. Provisional Application Ser. No. 61/920,070 filed on Dec. 23, 2013. All documents above are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2014/051266 | 12/23/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/095973 | 7/2/2015 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2869914 | Apr 2013 | CA |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20170038367 A1 | Feb 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61920070 | Dec 2013 | US |
