DETECTION OF PROTEIN TO PROTEIN INTERACTIONS
20220229068 · 2022-07-21
Assignee
Inventors
Cpc classification
C07K2319/92
CHEMISTRY; METALLURGY
G01N33/6845
PHYSICS
C12N15/1055
CHEMISTRY; METALLURGY
A01K67/0336
HUMAN NECESSITIES
G01N33/6803
PHYSICS
International classification
Abstract
A system and method for detecting interactions between a first protein or fragment thereof (bait protein) and a second protein or fragment thereof (prey protein) comprising: (a) a bait construct comprising the bait protein, a first epitope tag and an intein N-terminal fragment (IN); and (b) a prey construct comprising the prey protein, a second epitope tag, and an intein C-terminal fragment (IC).
Claims
1. An artificial split intein, wherein the artificial split intein comprises (i) a C-terminus fragment (IC) that includes amino acid residues at positions 13 to 37 of wild type IC of GP41-1, and a N-terminus fragment (IN) that includes amino acid residues at positions 1 to 88 of the wild type IN of GP41-1 fused to amino acid residues 1 to 12 of wild type IC of GP41-1 (C25 GP41-1 split intein), or (ii) an IC that includes amino acids at positions 14 to 37 of wild type IC of GP41-1 and an IN that includes amino acid residues at positions 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 13 of wild type IC of GP41-1 (C24 GP41-1 split intein), or (iii) an IC that includes amino acids at positions 15 to 37 of wild type IC of GP41-1 and an IN that includes amino acid residues at positions 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 14 of wild type IC of GP41-1 (C23 GP41-1 split intein).
2. A system for detecting interactions between a first protein or fragment thereof (bait protein) and a second protein or fragment thereof (prey protein) comprising: (a) a bait construct comprising the bait protein, a first epitope tag and an intein N-terminal fragment (IN); and (b) a prey construct comprising the prey protein, a second epitope tag, and an intein C-terminal fragment (IC), wherein (i) the IC includes amino acid residues at positions 13 to 37 of wild type IC of GP41-1, and the IN includes amino acid residues at positions 1 to 88 of the wild type IN of GP41-1 fused to amino acid residues 1 to 12 of wild type IC of GP41-1 (C25 GP41-1 split intein), or (ii) the IC includes amino acids at positions 14 to 37 of wild type IC of GP41-1 and the IN includes amino acid residues at positions 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 13 of wild type IC of GP41-1 (C24 GP41-1 split intein), or (iii) the IC includes amino acids at positions 15 to 37 of wild type IC of GP41-1 and the IN includes amino acid residues at positions 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 14 of wild type IC of GP41-1 (C23 GP41-1 split intein).
3. The system of claim 2, wherein the bait protein is fused at its C-terminus to the IN through the first epitope tag, and wherein the prey protein is fused at its N-terminus to the IC through the second epitope tag.
4. The system of claim 2, wherein the IN is fused to the N-terminal end of the bait protein while keeping the first epitope tag upstream (NIN), and the prey protein is fused at its N-terminus to the IC through the second epitope tag.
5. The system of claim 2, wherein the bait protein is fused at its C-terminus to the IN through the first epitope tag, and wherein the IC is fused to the C-terminal end of the prey protein while keeping the second epitope tag downstream (CIC).
6. The system of claim 2, wherein the bait construct further comprises a third epitope tag in tandem with the first tag.
7. The system of claim 6, wherein the first epitope tag, the second epitope tag and the third epitope tag include FLAG, V5-tag, Myc-tag, hemagglutinin (HA)-tag, Spot-tag and NE-tag.
8. (canceled)
9. The system of claim 2, wherein the bait protein is a soluble or membrane protein or fragment thereof.
10. The system of claim 2, wherein the prey protein is a soluble or membrane protein or fragment thereof.
11. A method for detecting the interaction between a first protein or part thereof (bait protein) and a second protein or part thereof (prey protein) comprising: (a) providing a bait construct as defined in claim 2; (b) providing a prey construct as defined in claim 2, wherein an association of the bait protein and the prey protein results in the IN and IC reconstituting into a functional intein molecule which then catalyzes its excision and formation of an intact protein which includes the first epitope tag and the second epitope tag; (c) incubating the bait construct and the prey construct under conditions that allow the formation of the intact protein to form an incubate; and (d) adding to the incubate a detectable agent that recognizes at least one of the first epitope tag and the second epitope tag to detect the formation of the intact protein, whereby detection of the intact protein being indicative that the first protein or part thereof and the second protein or part thereof interact.
12. The method of claim 11, wherein the detectable agent is an antibody.
13. The method of claim 11, wherein the method further comprises measuring an expression output of the detected intact protein as a measure of an amount of interaction between the first and the second proteins to quantitatively measure strength and affinity between the bait protein and the prey protein.
14. The method of claim 11, wherein the bait construct and the prey construct are expressed in a host cell.
15. The method of claim 14, wherein the bait construct and the prey construct are expressed in the host cell by: (i) introducing into the host cell as part of a bait vector, a first gene under the control of a promoter, said first gene coding inter alia for the bait protein which gene is attached to the DNA-sequence of a first module encoding inter alia the first epitope tag and the IN; and (ii) introducing into the host cell, as part of a prey vector, a second gene under the control of a promoter, the second gene coding inter alia for the prey protein which gene is attached to the DNA sequence of a second module encoding inter alia the second epitope tag and the IC.
16. The method of claim 15, wherein the bait vector is maintained episomally in the host cell or is integrated into the genome of the host cell.
17. The method of claim 15, wherein the prey vector is maintained episomally in the host cell or is integrated into the genome of the host cell.
18. The method of claim 12, wherein the bait construct further comprises a third epitope tag in tandem with the first epitope tag, and wherein the method further comprises performing another incubation of the incubate on a substrate coated with an anti-first epitope tag antibody for immunological immobilization of the bait construct to the substrate and wherein the detectable agent is an anti-third epitope tag antibody that allows for detection of the bait construct against either the first epitope tag or the third epitope tag.
19. The method according to claim 18, wherein the detection is performed as an ELISA assay or as a Homogeneous Time Resolved Fluorescence (HTRF) assay.
20. A method of identifying a potentially pharmaceutically active inhibitor of an interaction between a bait protein and a prey protein comprising: (a) providing a host cell; (b) expressing in the host cell the system of claim 2, the bait protein and the prey protein being selected such that they interact when expressed in the host cell, wherein the interaction of the bait protein and the prey protein results in the IN and IC reconstituting into a functional intein molecule which then catalyzes its excision and formation of an intact protein which includes the first epitope tag and the second epitope tag; (c) incubating the host cell in presence of a compound of interest under conditions that allow for the formation of the intact protein to form an incubate; and (e) adding to the incubate an antibody or antibodies that recognize at least one or both of the first epitope tag and the second epitope tag to detect the formation of the intact protein, wherein an absence of detection of the intact protein is indicative of the compound of interest being potentially pharmaceutically active inhibitor of the interaction between the prey protein and the bait protein.
21. A method for providing a compound that can interfere with protein/protein interaction, the method comprising: (a) providing host cells having the system of claim 2, the bait protein and the prey protein being selected such that they interact when expressed in the host cells to form an intact protein; (b) incubating a first set of the host cells under conditions that allow for expression of the intact protein in the presence of a compound of interest to obtain a first incubation and incubating a second set of the host cells under conditions that allow for expression of the intact protein in the absence of the compound of interest to obtain a second incubation; (c) measuring a level of expression of the intact protein in the first incubation and a level of expression of the intact protein in the second incubation, wherein when the level of expression of the intact protein in the first incubation is lower than the level of expression of the intact protein in the second incubation indicates that the compound interest interferes with protein/protein interaction, thereby providing the compound that can interfere with protein/protein interaction.
22. An isolated peptide comprising SEQ ID NO:3.
23. An isolated peptide comprising SEQ ID NO:4.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Embodiments will now be described, by way of example only, with reference to the drawings, in which:
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0052] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the meanings below. All numerical designations, e.g., dimensions and weight, including ranges, are approximations that typically may be varied (+) or (−) by increments of 0.1, 1.0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term “about”.
[0053] Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the terms “include”, “has” and their grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. “Consisting essentially of” when used to define systems, compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for using the split inteins of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
[0054] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range.
[0055] The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.
[0056] The term “IN” as used herein refers to the N-terminal portion of the intein protein.
[0057] The term “IC” is used to refer to the C-terminal portion of an intein protein.
[0058] “Bait” as used in this document is a test peptide or polypeptide or protein whose interaction to another peptide, polypeptide or protein (prey as defined below) is being studied.
[0059] The term “bait construct” or “bait fusion protein” as used in this document defines a fusion protein between a first test protein or bait peptide (bait), one or more other polypeptides, one of which is IN, and a tag (the tag in the bait fusion protein may be referred to as the “first tag”). The first tag may be located between the first test protein and the IN (bait-tag-IN) or at an end of the IN opposite to the end linked to the bait (i.e. bait-IN-tag).
[0060] The term “bait vector” as used in this document refers to a nucleic acid construct which contains sequences encoding the bait construct and regulatory sequences that are necessary for the transcription and translation of the encoded sequences by the host cell, and preferably regulatory sequences that are needed for the propagation of the nucleic acid construct in mammalian cells.
[0061] The term “prey construct” or “prey fusion protein” as used in this document defines a fusion between a second test peptide or prey peptide, one or more other polypeptides, one of which is IC, and a tag (the tag in the prey construct may be referred to as the “second tag”). The second tag may be located between the second test peptide and the IN (prey-tag-IC) or at an end of the IC opposite to the end linked to the prey (i.e. prey-IC-tag).
[0062] “Prey” as used in this document is a test peptide or polypeptide or protein whose interaction to another peptide, polypeptide or protein (bait, as defined above) is being studied.
[0063] The terms “prey vector” and “library vector” as used herein refer to a nucleic acid construct which contains sequences encoding the prey construct and regulatory sequences that are necessary for the transcription and translation of the encoded sequences encoding by the host cell.
[0064] The term “tag” as used in this document refers to a nucleic acid sequence or its translation product, which allows the immunological isolation, detection and/or purification of a polypeptide bound to the tag by means of an antibody directed specifically against the tag. Examples of tags that can be used in the present application include V5 tag, HA tag, 3×FLAG tag.
[0065] “Test polypeptide” is a polypeptide whose interaction with another polypeptide is being studied with SMPL.
[0066] By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
Overview
[0067] The present disclosure relates to a novel approach for protein-protein interaction (PPI) detection that enables a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, a split intein is used as a sensor for protein interactions. The present disclosure enables in situ analysis of interactions occurring in various cellular compartments as well as their responses to pharmacological challenges such as enzymatic and PPI inhibitors.
[0068] Bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, a SIMPL ELISA platform is disclosed that enables high-throughput screening of PPIs and their inhibitors.
[0069] In one embodiment, the re-engineered or artificial split intein of the present disclosure comprises a C-terminus fragment (IC) that includes amino acid residues at positions 13 to 37 of wild type IC of GP41-1, and a N-terminus fragment (IN) that includes amino acid residues at positions 1 to 88 of the wild type IN of GP41-1 fused to amino acid residues 1 to 12 of wild type IC of GP41-1 (C25 GP41-1 split intein), or (ii) the IC includes amino acids at positions 14 to 37 of wild type IC of GP41-1 and amino acid residues at positons 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 13 of wild type IC of GP41-1 (C24 GP41-1 split intein), or (iii) the IC includes amino acids at positions 15 to 37 of wild type IC of GP41-1 and amino acid residues at positons 1 to 88 of wild type IN of GP41-1 fused to amino acid residues at positions 1 to 14 of wild type IC of GP41-1 (C23 GP41-1 split intein).
SIMPL
[0070] The SIMPL design includes (a) a bait construct or bait fusion protein carrying a bait, and (b) prey construct or a prey fusion protein carrying a prey.
[0071] To investigate the interaction of the test proteins in vivo both the bait fusion protein and the prey fusion protein are expressed in a cell line of interest, including mammalian and non-mammalian cells, preferably mammalian cells. If the bait and the prey interact, then the association of the bait in the bait fusion protein and the prey in the prey fusion protein brings IN and IC into close proximity, allowing them to reconstitute into a fully functional intein, which then catalyzes its excision and the concurrent ligation of the bait and the prey (as well as their respective tags) into an intact protein. The resulting spliced protein can be resolved by regular analytical procedures such as Western blot analysis due to its altered mobility, while the presence of the tags allows one or more of visualization, isolation, immobilization or purification of the intact protein using regular biochemical techniques.
[0072] In one embodiment, the bait is connected to the IN at its N-terminus directly, or indirectly through the first tag (see
[0073] In one embodiment, the prey protein is connected to the IC at its N-terminus directly, or indirectly through the second tag (see
[0074]
[0075] The following is a list of the intact proteins that result from the different combinations of bait construct and prey construct: [0076] IN+IC: intact peptide includes bait-first tag-second tag-prey (see
[0079] In one embodiment, the disclosure also provides for a CIC-GFP construct (prey-IC-FLAG (second tag)-GFP; see
HI Throughput Constructs
[0080] Use of the SIMPL construct of the present disclosure in combination with an analytical procedure such as Western blot analysis is applicable to detailed PPI analysis, this analysis is limited to low throughput analyses. Western blot analysis, although quantifiable, is not a preferred analytical method to quantify PPI. As such, the present disclosure provides, in another embodiment, for SIMPL constructs that can be used in ELISA for high-throughput, quantifiable measurements of PPI.
[0081] In this embodiment, the bait construct may include two different tags in tandem (see
[0082] The constructs and methods of the present disclosure provide for detection of physiological PPIs (see
Applications
[0083] The SIMPL constructs of the present disclosure may be used as a high-throughput screening technology for the identification of PPI of any proteins.
[0084] In addition SIMPL is sensitive enough to detect subtle changes in protein interactions, which can differ slightly depending on the presence or absence of various stimuli, like hormones or agonists, or inhibitory drugs. Specifically, SIMPL follows the kinetic process of kinase/substrate interactions.
(2) Drug Screening Platform
[0085] SIMPL can be used as a drug screening platform suitable for the identification of small molecule inhibitors or enhancers that alter a defined set of membrane protein interactions in their natural environment.
[0086] In another embodiment, the present disclosure provides for a kit of reagents for detecting binding between a first protein (membrane or soluble) or part thereof and a second protein or part thereof (membrane bound or soluble). The kit, in one embodiment, may include: (a) a host cell; (b) a first bait vector (bait), which may be maintained episomally or integrated into the genome of the host cell, comprising a first nucleic acid coding for a bait protein or part thereof, an IN and a first tag, the first bait vector may further include a promoter; (c) a second vector (prey), which may be maintained episomally or integrated into the genome of the host cell, comprising a second nucleic acid coding for a prey protein or part thereof, an IC and a second tag, the second prey vector may further comprise a promoter. In one aspect, the kit further includes (d) a plasmid library encoding second proteins or parts thereof.
Advantages
[0087] The system of the present disclosure presents several advantages over existing techniques to study interactors of membrane proteins: (i) SIMPL can be carried out in virtually any cell line due to the availability of prey/bait/reporter vectors for lentivirus generation, which poses the advantage of single copy integration and diminishes overexpression artifacts. Moreover, SIMPL is carried out in living cells, thus avoiding signal changes arising from cell lysis or protein purification used in biochemical PPI methods; (ii) SIMPL is compatible with ELISA, allowing for a fast, high throughput and quantifiable results; (iii) SIMPL can detect subtle changes in interaction patterns, which can be induced/repressed by either drugs, various stimuli or phosphorylation events, in a highly specific manner; (iv) SIMPL can be used as a platform for drug discovery, specifically used to screen for novel compounds capable of inhibiting signaling mediated by oncogenic receptors; (vi) SIMPL may be used in quantitative studies to measure the strength or affinity of PPI; (vii) as splicing occurs in situ, both loss of specific interaction and gain of nonspecific interaction during processing steps, which are common problems for many affinity-based methods such as co-immunoprecipitation and AP-MS, are avoided; and SIMPL can detect PPIs in various cellular compartments; (viii) SIMPL can be coupled to Homogeneous Time Resolved Fluorescence (HTRF).
[0088] In order to aid in the understanding and preparation of the present disclosure, the following illustrative, non-limiting examples are provided.
Examples
Materials and Methods
[0089] Molecular cloning and library preparation. The plasmids containing GP41-1 split intein cDNA, pCAG-Co-InCreN and pCAG-Co-InCreC, were obtained from Addgene. SIMPL bait and prey vectors are generated by integrating DNA pieces of GP41-1 split intein fragments, linkers, and tags, as well as Gateway cloning cassette, into pCMV5 vector backbone by Gibson assembly (New England BioLabs). Plasmids for FlpIn stable cloning were created similarly into pCDNA5/FRT/TO vector with both bait and prey included by Gibson assembly. Most cDNAs were originally obtained from human ORFeome collection or from the Openfreezer collection at Lunenfeld-Tanenbaum Research Institute [39]. Those not in entry clone vectors were cloned into pDONR223 by PCR and Gateway BP reactions (Life Technologies). Different cDNA fragments were then cloned into SIMPL vectors by Gateway LR reactions (Life Technologies). Site-directed mutagenesis was generated by PCR using KAPA HiFi DNA polymerase (KAPA Biosystems). The plasmids created in this study are available from the corresponding author upon reasonable request.
[0090] The natural version (WT) of intein N-terminal fragment (IN) of GP41-1 split intein contains 88 amino acids. Its correspondent (WT) intein C-terminal fragment (IC) contains 37 aa (see
[0091] Cell culture and treatment. HEK 293 and HeLa cell lines were generous gifts from Dr. J. Moffat. HEK 293 Flp-In T-Rex cell line was a generous gift from Dr. A. C. Gingras. These cells were grown in DMEM supplemented with 10% fetal calf serum (Life Technologies). PC9 cell line was a generous gift from Dr. P. Jannes and they were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. For ELISA assay, cells were seeded in 96 well (Sarstedt AG & Co) or 384 well plates (Greiner Bio-One) with 15,000 (96 well) or 5,000 (384 well) cells per well. Cells were transfected with various plasmids with polyethylenimine (PEI) Max (Polysciences) [40]. Expression in HEK 293 Flp-In T-Rex cells was induced by treating the cells with tetracycline (1 μg/ml) for 6-16 hrs. For experiments to study signaling pathway activation, the cells were starved with 0.1% fetal calf serum as well as treated with tetracycline for 6 hrs before stimulation with EGF (Sigma-Aldrich), Tetradecanoylphorbol acetate (TPA) (Sigma-Aldrich) or anisomycin (Sigma-Aldrich). Stable cell lines were created according to the manual of Flp-In T-REx (Invitrogen). Briefly, plasmid containing both bait and prey DNA and pOG44 plasmid (1:10 ratio) were cotransfected into HEK 293 Flp-In T-Rex cells.
[0092] After 3 days, the cells underwent puromycin selection. Single colonies were selected and the expression of bait and prey were verified by Western blot analysis.
[0093] Western blot analysis and immunoprecipitation. Cells were lysed in buffer H (Triton X 100 1%, β-glycerophosphate pH7.3 50 mM, EGTA 1.5 mM, EDTA 1 mM, orthovanadate 0.1 mM, DTT 1 mM supplemented with protease inhibitors (Roche)). After centrifugation at 21,000×g for 10 min, the supernatants were mixed with Laemmli sample buffer, boiled at 95° C. for 3-5 min and subjected to Western blot analysis. For immunoprecipitations, supernatants (0.3 ml) were incubated with antibodies at 4° C. with rotation for one hour, followed by another hour of incubation with protein G sepharose beads (GE Healthcare). Beads were washed twice with LiCl (0.5 M in Tris pH8.0 0.1 mM) and twice with lysis buffer, boiled with Laemmli sample buffer and then were subjected to Western blot analysis. Antibodies used for Western blot analysis and immunoprecipitations were: α-FLAG antibody purchased from Sigma-Aldrich Co. (F1804) with 1:10,000 dilution and α-V5 antibody from Cell Signaling Technology (#13202) with 1:10,000 dilution. Each of the above antibodies was diluted according to provider's protocol.
[0094] ELISA. HEK 293 cells were grown in 96 well or 384 well plates and were transfected with PEI as aforementioned. The cells in each well were lysed in 120 μl (96 well) or 80 μl (384 well) THE buffer (Tris pH7.5 20 mM, NaCl 150 mM, EDTA 2 mM and Triton X-100 0.5% supplemented with protease inhibitors). Aliquots of lysates (20 μl) were incubated for 3 hrs at 40 C in a well of a 384 well Lumitrac plate (Greiner Bio-One) that was coated with α-FLAG antibody (20 μl/well with 1:100 dilution) and blocked with BSA. After 3 times thorough wash with phosphate buffer saline supplemented with 0.05% Tween 20 (PBST), the plate was incubated with HRP-conjugated α-HA antibody (GeneTex GTX115044, 1:5,000 dilution) for 1 hr at room temperature. The plate was washed 3 times with PBST followed by chemiluminescence reading using SuperSignal ELISA Pico substrate (ThermoFisher).
[0095] Selection of RRS pairs. All bait-prey pairs (75 baits×78 preys) were considered for the RRS, and 88 were selected that had the lowest chances of interaction, using the following criteria: 1. Absence from the PRS; 2. Absence from the Integrated Interactions Database ver. 2018-11 [41], thereby ensuring that the pairs had not been detected in experimental studies, predicted based on orthology, or predicted by five computational algorithms; 3. Lowest probabilities of interaction according the FpClass PPI prediction algorithm [42]; 4. Maximal coverage of candidate baits and preys.
[0096] C. elegans SIMPL vectors and cloning. To facilitate assembly of the expression plasmids, we used a SapI-based cloning strategy. We generated a series of donor vectors based on the kanamycin resistant cloning vector pHSG298 (Takara Bio), in which the insert is flanked with SapI-sites. Digestion with SapI yields overhangs that enable assembly of promoter, ORF, split-intein, and UTR into a destination vector (pMLS257 Addgene #73716) in a single ligation reaction. The following plasmids were generated: i) donor plasmids containing IN (pJRK244), IC (pJRK036), and CIC (pJRK152) split-intein donor sequences. Split-intein amino-acid sequences are identical to the mammalian ELISA compatible split-intein constructs, but are codon optimized for C. elegans and contain an artificial intron. ii) two rps-0 promoter donor plasmids, pJRK001 for assembly with IC and IN, and pJRK151 for assembly with CIC. iii) three unc-54 3′-UTR plasmids: pJRK150 for assembly with IC, pJRK153 for assembly with CIC, and pJRK002 for assembly with IN. ORFs were amplified by PCR from a mixed-stage cDNA library and cloned blunt-ended into vector pHSG298 digested with Eco53kl. Plasmid sequences available upon request. Plasmids used for injection were purified using the PureLink HQ Mini Plasmid DNA Purification Kit (ThermoFisher) using the extra wash step and buffer recommended for endA+ strains.
[0097] C. elegans strain and culture conditions. C. elegans strains were cultured under standard conditions (Brenner, S. The genetics of Caenorhabditis elegans. Genetcis 77, 71-94 (1974)). Only hermaphrodites were used and all experiments were performed with animals grown at 20° C. on Nematode Growth Medium (NGM) agar plates seeded with E. coli OP50 bacteria.
[0098] Extrachromosomal strain generation. Young adult N2 animals were injected with 20 ng/μl of the prey IC/CIC SIMPL plasmid, 5 ng/μl of the bait IN SIMPL plasmid, 20 ng/μl of a plasmid conferring a dominant RoI phenotype and Hygromycin B resistance (pDD382 Addgene #91830), and 55 ng/μl lambda DNA (ThermoScientific SM0191). Four hermaphrodites were injected for each protein-pair and placed on individual plates. After 2-3 days, Hygromycin B (250 μg/ml) was added to the plates to select for transgenic lines. From each plate a single F2 RoI animal was picked to establish up to four transgenic strains per protein pair, and each was tested for expression of the SIMPL constructs.
[0099] C. elegans lysis and ELISA. Mixed-stage animals grown under Hygromycin B (250 μg/ml) selection were washed off with M9 buffer (0.22 M KH2PO4, 0.42 M Na2HPO4, 0.85 M NaCl, 0.001 M MgSO4), and washed two more times with M9 buffer. Samples were then pelleted and resuspended in 100-400 μl of Lysis Buffer (25 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Igepal 630, and 1 tablet/50 ml cOmplete protease inhibitor cocktail (Sigma-Aldrich)). After flash freezing in liquid nitrogen and thawing, samples were sonicated with a Diagenode BioRupter Plus, 5 min high setting: 30 s on/30 s off in a 4° C. water bath. The lysates were then spun at max speed in a tabletop centrifuge at 4° C. for 15 min to clear cellular debris. ELISA was performed as above, but the SuperSignal ELISA pico chemiluminescent substrate was used undiluted (ThermoScientific).
[0100] Statistics. Two-tailed Student's t-test with n=3 was used to examine the significance of kinase/substrate and mitochondrial PPIs.
Results
Design and Facilitation of SIMPL
[0101] In one SIMPL design (
[0102] The GP41-1 split intein, which was identified from environmental metagenomics sequence data [9], was chosen for use in the SIMPL system due to its small size (88 amino acids long in IN and 37 amino acids long in IC) and because it possesses the most rapid reaction rate among all split inteins examined [7,10]. Rapamycin-induced heterodimerization of FKBP1A (FKBP12) (IC fused) and FKBP rapamycin-binding (FRB) domain of mTOR11 (IN fused) was used as a test case to evaluate SIMPL performance in a HEK 293 mammalian cell background. Using the WT form of GP41-1, which is split at position C37 (numbered from the last C-terminal amino acid of IC), led to a relatively high level of FRB/FKBP1A splicing even in the absence of rapamycin treatment (
[0103] To further characterize the SIMPL system, we treated the HEK 293 cells expressing FRB/FKBP1A SIMPL constructs with different concentrations of rapamycin (
[0104] We further assessed SIMPL in isogenic stable cells to examine potential problems derived from transient transfection such as uneven expression in various cells and difficulty in manipulating expression level. The stable cell line was created by incorporating both FRB-IN and IC-FKBP1A into the genome of host Flp-In T-Rex HEK 293 cells through Flp recombinase-mediated integration. Cells with different expression levels of both FRB and FKBP1A, induced by incubation with varied doses of tetracycline, were treated with rapamycin (
[0105] Alternative SIMPL formats to extend its detection capability. In the above prototypic SIMPL design, a bait molecule is fused at its C-terminus to the IN and a prey is fused at its N-terminus to the IC. While functional in many cases, this strict arrangement limits the overall detection capability of SIMPL because in some instances the two tags in this format may be spatially inaccessible to each other. Additionally, the function of some proteins may be disrupted by the presence of tags on specific termini, necessitating a different strategy. To address these limitations, we designed alternative intein construct arrangements (
[0106] It should be noted that a basal splicing signal appeared in the sample of NIN/CIC-GFP combination without rapamycin treatment. This might be derived from an affinity change caused by different tagging or high-level expression of the proteins. However, the corresponding rapamycin-treated sample shows a dramatically increased signal (more than eight fold by density), making the states easily distinguishable from one other when proper controls and quantification are used. Thus, all four combinations are suitable for use in PPI detection in SIMPL.
[0107] ELISA platform of SIMPL assay and its unbiased evaluation. While use of SIMPL with a Western blot readout is applicable to detailed PPI analysis, it is limited to low-throughput analyses and is not strongly quantifiable. We therefore developed an alternative, ELISA-coupled SIMPL platform for high-throughput, quantifiable measurement of PPIs. For this purpose, a hemagglutinin (HA) tag was introduced into the bait construct in tandem with V5. This allows for monitoring of protein splicing using an ELISA format, with protein capture performed using α-FLAG antibody and detection performed using α-HA antibody coupled to horseradish peroxidase (HRP) (
[0108] We next evaluated the SIMPL system using a benchmarking approach with unbiased PPI reference sets, which has been widely accepted for assessing the overall performance of a PPI method. We employed a positive reference set (PRS) which contained 88 available positive PPIs derived from the previously well-established human PRS (hPRS)15, including different types of PPIs and covering those occurring in various subcellular locations (Table 2). Our random reference set (RRS) contained 88 protein pairs with baits and preys selected from the PRS but used in combinations determined computationally to have low probability of interaction (Table 2). The reference sets were evaluated with the ELISA platform in two formats, bait(IN)/prey(IC) and bait(IN)/prey(CIC) (Table 3). The expression of baits in the same samples was also tested with ELISA (
Further Characterization of SIMPL with PPIs Involved in Signaling Pathways and Mitochondrial Biogenesis.
[0109] We next used SIMPL to explore physiological PPIs and chose PPIs in the EGFR-RAS-ERK1/2 axis, an important signaling pathway involved in multiple physiological and pathological processes [18]. Activated EGFR undergoes autophosphorylation on tyrosine residues, which recruit scaffold proteins such as SHC1 to relay signal to downstream machinery [19]. At the RAS level, activated RAS (KRAS in this study) binds and directly activates RAF kinases20 (RAF1 in this study). When EGFR-IN and IC-SHC1 were co-expressed in HEK 293 cells, their interaction was captured as a spliced band above EGFR recognizable by both α-FLAG and α-V5 antibodies (
[0110] As the study of rapamycin-induced FRB/FKBP1A interaction demonstrated the potential of SIMPL to follow interaction kinetics (
[0111] As most PPI methods are not well suited for detection of weak or transient PPIs, we wanted to test whether SIMPL is capable of following these types of interactions. We selected protein kinases as our test case since their association with substrates, similar to common enzyme/substrate interactions, is usually characterized as transient and weak [24]. Thus, IN-fused MAPK1 (ERK2), MAPK8 (JNK1), MAPK14 (p38α MAPK), MAPK7 (ERK5), AKT1 and PRKCA (PKCα) were examined with several well documented substrates in both IC and CIC formats. ELISA assay demonstrated that more than 50% of these PPIs could be detected by SIMPL, in one or both formats (
[0112] The evaluation with the reference PPI set suggests SIMPL is capable of detecting PPIs in various cellular compartments as the PRS covers PPIs occurring in different locations such as nucleus, cytoplasm, plasma membrane and extracellular space (Table 2). We further tested this by examining mitochondrial PPIs with SIMPL as mitochondria are special organelles with distinct features and their PPIs are often difficult to study. We selected several well-studied mitochondrial PPIs for this purpose (Table 4), including proteins involved in oxidative phosphorylation [26], transport [27], cristea organization [28] and metabolism [29]. Baits were prepared using the IN format to avoid interference of transit peptides usually at N-termini of mitochondrial proteins. The corresponding preys were constructed in either the IC or CIC configuration (or both), to reduce the chance of steric interference preventing their association with IN or to prevent incorrect sorting of the prey proteins. Out of 10 PPIs examined, eight (TIMM50/TIMM23, PDHA1/PDHB, CHCHD6/CHCHD3, NDUFV1/NDUFV3, SDHA/SDHB, UQCRC2/UQCRC2, ATP5MC1/ATP5MC1 and ETFA/ETFB) were successfully detected (
[0113] We tested two additional controls for PDHA1/PDHB interaction to exclude the possibility that splicing occurred in cell lysates during sample processing (
[0114] These results display the broad applicability of SIMPL for monitoring interactions with diverse subcellular localizations.
Detecting PPIs in C. elegans with SIMPL
[0115] We next investigated the feasibility of using the SIMPL system in a multicellular animal using the nematode Caenorhabditis elegans as a model. We selected a C. elegans PRS of 27 PPI pairs from previously identified literature confirmed interactions [43] and from interactions used previously to evaluate binary PPI mapping approaches [44] (Table 5). We also assembled a C. elegans RRS by randomly combining protein pairs from the PRS, excluding known interactors. Full-length ORFs of the corresponding genes were PCR amplified and cloned into vectors containing split-intein tags optimized for expression in C. elegans, but otherwise identical to the ELISA compatible split-intein fragments used above.
[0116] Proteins were expressed under control of the general rps-0 ribosomal promoter. Transgenic animals were generated by microinjection, and both IC and CIC configurations were injected for each prey protein. To enable accurate quantification of splicing by ELISA, we injected a 4× higher concentration of prey plasmid than bait plasmid to ensure that splicing of the bait protein is not limited by the availability of the prey protein. All transgenic lines were first tested for expression of both bait and prey protein by Western blotting. In all, we recovered 10 PRS pair-expressing lines and 13 RRS pair-expressing lines, representing 7 and 9 unique protein pairs respectively (
SIMPL as a Drug Screening Platform.
[0117] We investigated whether SIMPL can serve as a drug screening tool, in particular as an assay that could detect enzymatic- as well as PPI-inhibitors. Since protein splicing is an irreversible process, inhibitors have to be administered before bait/prey expression (
Development of Screen System for Protein-Protein Interaction (PPI) Inhibitor
[0118] Previously we have shown that SIMPL can serve as an assay to verify activity of different PPI inhibitors. However, this demonstration was carried out by expressing the targeted proteins transiently in the cells, and testing activity of EGFR inhibitor AG1478 and BCL2 inhibitor Veneto lax. The transient ectopic expression restricts the system from being applicable to large scale screening. To overcome this limitation, we created stable cell lines by integrating target genes into the genome of the host cells. This is showcased using KRAS. More specifically, the expression plasmid with open reading frames of both KRAS and Ras binding domain (RBD, derived from Raf1 and responsible for binding to Ras) under the control of T-Rex promoter was created (
SIMPL Coupled to HTRF Assay
[0119] ELISA is currently used as the readout for medium to high throughput SIMPL assay. However, the tedious procedures that accompany use of ELISA make it less satisfactory. We decided to improve the performance of our assays by coupling SIMPL with Homogeneous Time Resolved Fluorescence (HTRF). In this assay, cell lysates are incubated with antibodies conjugated with fluorescent dyes (Tb as fluorescence donor and d2 as acceptor). After incubation, the splicing signal is read as delayed Fluorescence Resonance Energy Transfer (FRET). Since the emission of the donor dye is relatively sustainable, the real FRET signal can be obtained by delayed measurement and the background signals are filtered out due to their fast emission. The feasibility of HTRF readout platform was demonstrated using Rapamycin-induced FRB/FKBP1A interaction (
[0120] Next, we evaluated the HTRF-coupled system using AMG510 to inhibit KRAS(G120)/RBD interaction. The cells with stable integration of KRAS (WT and mutants) and RBD genes (as described in the previous section) were treated with both Tetracycline and AMG510 overnight followed by HTRF assay. The inhibition of KRAS/RBD interaction by AMG510 was only observed in G12C mutant, and not in WT nor G12V mutant. We therefore conclude that HTRF can be used as a powerful readout platform for SIMPL.
TABLE-US-00001 TABLE 2 Reference sets for evaluating SIMPL assay (a) Positive reference set (PRS) bait prey protein subcellular protein subcellular name localization name localization AKT1 cell membrane PDPK1 plasma membrane (associated), (peripheral), cytoplasm, nucleus nucleus, cytosol AKT1 cell membrane TCL1A nucleus, ER (associated), ARFIP2 plasma membrane, cytoplasm, nucleus cytosol, ARF1 Golgi apparatus Golgi apparatus (anchored), DDIT3 nucleus plasma membrane HLA-A plasma membrane, (anchored) ER, Golgi apparatus ATF3 nucleus HLA-B plasma membrane, B2M extracellular ER, Golgi apparatus B2M extracellular HLA-C plasma membrane, B2M extracellular ER, Golgi apparatus BAD mitochondrion (outer BCL2L1 mitochondrion, membrane), cytoplasm cytoskeleton, BAK1 mitochondrion (outer nucleus, cytosol membrane) BCL2L1 mitochondrion, BDNF extracellular cytoskeleton, CASP2 cytosol, nucleus, cytosol mitochondrion, NTF4 extracellular nucleus CRADD nucleus, cytosol CBLB cytoplasm GRB2 plasma membrane, CCND3 nucleus cytoplasm, nucleus CD2 cell membrane CDK6 nucleus, cytoskeleton, CDK2 nucleus, cytoplasm, cytosol cytoskeleton, CD58 plasma membrane endosome CKS1B nucleus CDKN1A nucleus CCNA1 nucleus CDKN1B nucleus, endosome CCNA1 nucleus CEBPG nucleus FOS nucleus, ER, cytosol CGA extracellular CGB5 exracellular CRK cytoplasm, nucleus, PDGFRB plasma membrane, membrane (associated) lysosome CXCL1 extracellular CXCR2 plasma membrane DDIT3 nucleus FOS nucleus, ER, cytosol DR1 nucleus DRAP1 nucleus ERBB3 plasma membrane NRG1 membrane, FABP5 nucleus, cytosol, extracellular membrane, S100A7 extracellular, extracellular cytoplasm, nucleus FEN1 nucleus PCNA nucleus FGF1 extracellular, cytosol, FGFR1 plasma membrane nucleus PCNA nucleus GADD45A nucleus LAT plasma membrane GRAP2 endosome, cytosol, LAT plasma membrane nucleus PTK2 plasma membrane GRB2 plasma membrane, (associated), cytoplasm, nucleus cytoskeleton, nucleus, GRB2 plasma membrane, cytosol cytoplasm, nucleus VAV1 cytosol, plasma GRB2 plasma membrane, membrane cytoplasm, nucleus GTF2F2 nucleus GTF2F1 nucleus HBB cytosol HBA2 cytosol RB1 nucleus HDAC1 nucleus ZBTB16 nucleus HDAC1 nucleus TP53 nucleus, HIF1A nucleus, cytosol mitochondrion, IFIT1 cytoplasm ER IGF2 extracellular EIF3E nucleus, cytosol JUNB nucleus IGFBP4 extracellular LCP2 cytoplasm BATF nucleus LCP2 cytoplasm GRAP2 endosome, cytosol, LCP2 cytoplasm nucleus LGALS3 extracellular, plasma NCK1 plasma membrane, membrane, nucleus, ER, nucleus, cytosol cytoplasm VAV1 cytosol, plasma LMNA nucleus membrane LMNA nucleus LGALS3BP extracellular, nucleus, LSM3 nucleus plasma membrane MAD2L1 nucleus, cytoskeleton, LMNB1 nucleus kinetochore RB1 nucleus MAFG nucleus LSM2 nucleus MAPK7 nucleus, cytoplasm MAD1L1 cytoskeleton, nucleus, MCM2 nucleus kinetochore MCM2 nucleus NFE2L1 ER, nucleus NCBP1 nucleus MAP2K5 nucleus, cytoplasm NF2 plasma membrane MCM3 nucleus (associated), nucleus MCM5 nucleus, cytosol NR3C1 mitochondrion, NCBP2 nucleus cytosol, nucleus, HGS endosome, cytoplasm cytoskeleton HSP90AA1 plasma membrane, NR3C1 mitochondrion, cytoplasm, nucleus cytosol, nucleus, RELA nucleus, cytoplasm cytoskeleton MCM10 nucleus ORC2 nucleus ORC4 nucleus ORC2 nucleus RACK1 plasma membrane, PDE4D plasma membrane nucleus, (associated), mitochondrion, cytoskeleton cytosol PDGFRB plasma membrane, PTPN11 nucleus, cytoplasm lysosome PEX19 peroxisome PEX14 peroxisome PEX11B peroxisome PEX19 peroxisome PEX16 peroxisome PEX19 peroxisome PEX3 peroxisome PEX19 peroxisome PPP3R1 cytosol, plasma PPP3CA nucleus, cytosol, membrane plasma membrane (anchored), (peripheral) nucleus PRKAR2A plasma membrane, EZR plasma membrane cytoplasm, nucleus (peripheral), PSMD4 cytosol, nucleus cytoskeleton, nucleus, PTK2 plasma membrane cytosol, endosome (associated), RAD23A nucleus, cytosol cytoskeleton, nucleus, SRC plasma membrane, cytosol cytoskeleton, PTPN11 nucleus, cytoplasm cytoplasm, nucleus, RAC1 plasma membrane mitochondrion (anchored), cytoplasm FRS2 plasma membrane, RAF1 cytosol, plasma cytosol, endosome membrane, nucleus ARFIP2 plasma membrane, RCC1 nucleus cytosol, RET plasma membrane, Golgi apparatus endosome RAP1A plasma membrane RHOA cytoskeleton, plasma (anchored), endosome membrane (anchored) RAN nucleus, cytosol RIPK2 cytoplasm FRS2 plasma membrane, RPA2 nucleus cytosol, endosome S100A1 nucleus, cytoplasm ARHGAP1 cytoplasm S100A6 plasma membrane NOD1 plasma membrane, (peripheral), nucleus, cytoplasm cytoplasm RPA3 nucleus SKP1 cytoplasm, nucleus S100B nucleus, cytoplasm SKP1 cytoplasm, nucleus S100B nucleus, cytoplasm SMAD1 nucleus, cytoplasm BTRC nucleus, cytosol SMAD3 nucleus, cytoplasm SKP2 nucleus, cytoplasm SMAD4 nucleus, cytoplasm SMAD4 nucleus, cytoplasm TNFSF10 extracellular, plasma SMAD4 nucleus, cytoplasm membrane DCP1A nucleus TP53 nucleus, TNFRSF10B plasma membrane mitochondrion, UBE2I nucleus, cytosol ER CASP3 cytoplasm, nucleus XIAP nucleus, cytoplasm CASP7 cytoplasm, nucleus XIAP nucleus, cytoplasm CASP9 cytosol, XIAP nucleus, cytoplasm mitochondrion Note: the “Prey” column should be read next to the “Bait” column.
TABLE-US-00002 TABLE 2 (b) Random reference set bait prey subcellular subcellular name localization name localization AKT1 cell membrane PEX19 peroxisome (associated), SKP2 nucleus, cytoplasm cytoplasm, nucleus LMNB1 nucleus ARF1 Golgi apparatus PEX16 peroxisome (anchored), plasma RAD23A nucleus, cytosol membrane (anchored) TP53 nucleus, ATF3 nucleus mitochondrion, B2M extracellular ER B2M extracellular DCP1A nucleus B2M extracellular RAP1A plasma membrane BAD mitochondrion (anchored), endosome (outer membrane), MAD1L1 cytoskeleton, nucleus, cytoplasm kinetochore BAK1 mitochondrion MCM5 nucleus, cytosol (outer membrane) VAV1 cytosol, plasma BDNF extracellular membrane BDNF extracellular BCL2L1 mitochondrion, CASP2 cytosol, cytoskeleton, nucleus, mitochondrion, cytosol nucleus GTF2F2 nucleus CBLB cytoplasm HGS endosome, cytoplasm CD2 cell membrane DDIT3 nucleus CD2 cell membrane PEX11B peroxisome CDK2 nucleus, cytoplasm, DRAP1 nucleus cytoskeleton, ARHGAP1 cytoplasm endosome CASP9 cytosol, CDKN1A nucleus mitochondrion CDKN1B nucleus, endosome CD58 plasma membrane CEBPG nucleus MCM3 nucleus CEBPG nucleus NCBP2 nucleus CGA extracellular UBE2I nucleus, cytosol CGA extracellular MAP2K5 nucleus, cytoplasm CGA extracellular ORC4 nucleus CRK cytoplasm, nucleus, SMAD4 nucleus, cytoplasm membrane (associated) NRG1 membrane, CXCL1 extracellular extracellular CXCL1 extracellular NTF4 extracellular CXCLI extracellular DDIT3 nucleus DDIT3 nucleus PCNA nucleus DR1 nucleus PTK2 plasma membrane ERBB3 plasma membrane (associated), ERBB3 plasma membrane cytoskeleton, nucleus, FABP5 nucleus, cytosol, cytosol membrane, GRAP2 endosome, cytosol, extracellular nucleus FEN1 nucleus NCK1 plasma membrane, FGF1 extracellular, cytosol, ER, nucleus, cytosol nucleus RB1 nucleus FGF1 extracellular, cytosol, FRS2 plasma membrane, nucleus cytosol, endosome GADD45A nucleus PDPK1 plasma membrane GADD45A nucleus (peripheral), nucleus, GRAP2 endosome, cytosol, cytosol nucleus NFE2L1 ER, nucleus GRB2 plasma membrane, PEX3 peroxisome cytoplasm, nucleus NOD1 plasma membrane, GTF2F1 nucleus cytoplasm HBA2 cytosol CKS1B nucleus HDAC1 nucleus LAT plasma membrane HDAC1 nucleus PPP3R1 cytosol, plasma HIF1A nucleus, cytosol membrane IFIT1 cytoplasm (anchored), IGF2 extracellular nucleus IGF2 extracellular ARFIP2 plasma membrane, IGF2 extracellular cytosol, Golgi JUNB nucleus apparatus LCP2 cytoplasm RELA nucleus, cytoplasm LGALS3 extracellular, plasma ARFIP2 plasma membrane, membrane, nucleus, cytosol, Golgi cytoplasm apparatus LMNA nucleus HSP90AA1 plasma membrane, LSM3 nucleus cytoplasm, nucleus LSM3 nucleus RPA3 nucleus MAD2L1 nucleus, cytoskeleton, PTPN11 nucleus, cytoplasm kinetochore CRADD nucleus, cytosol MAD2L1 nucleus, cytoskeleton, LSM2 nucleus kinetochore PEX3 peroxisome MAFG nucleus FGFR1 plasma membrane MAFG nucleus LGALS3BP extracellular, nucleus, MAPK7 nucleus, cytoplasm plasma membrane MCM2 nucleus NFE2L1 ER, nucleus NCBP1 nucleus NTF4 extracellular NF2 plasma membrane PEX11B peroxisome (associated), nucleus TNFRSF10B plasma membrane NR3C1 mitochondrion, PEX16 peroxisome cytosol, nucleus, S100B nucleus, cytoplasm cytoskeleton S100B nucleus, cytoplasm ORC2 nucleus FOS nucleus, ER, cytosol ORC2 nucleus RAD23A nucleus, cytosol PDE4D plasma membrane HBB cytosol (associated), UBE2I nucleus, cytosol cytoskeleton EZR plasma membrane PDGFRB plasma membrane, (peripheral), lysosome cytoskeleton, PEX14 peroxisome nucleus, cytosol, PEX19 peroxisome endosome PPP3CA nucleus, cytosol, RAN nucleus, cytosol plasma membrane NRG1 membrane, (peripheral) extracellular PRKAR2A plasma membrane, BATF nucleus cytoplasm, nucleus FOS nucleus, ER, cytosol PSMD4 cytosol, nucleus SRC plasma membrane, PSMD4 cytosol, nucleus cytoskeleton, RAC1 plasma membrane cytoplasm, nucleus, (anchored), cytoplasm mitochondrion RCC1 nucleus NOD1 plasma membrane, RET plasma membrane, cytoplasm endosome TCL1A nucleus, ER RHOA cytoskeleton, plasma BTRC nucleus, cytosol membrane (anchored) CASP7 cytoplasm, nucleus RHOA cytoskeleton, plasma NCBP2 nucleus membrane (anchored) CCNA1 nucleus RIPK2 cytoplasm ZBTB16 nucleus RIPK2 cytoplasm IGFBP4 extracellular RPA2 nucleus PDGFR8 plasma membrane, S100A1 nucleus, cytoplasm lysosome S100A6 plasma membrane S100A7 extracellular, (peripheral), nucleus, cytoplasm, nucleus cytoplasm CASP3 cytoplasm, nucleus SKP1 cytoplasm, nucleus RAN nucleus, cytosol SMAD1 nucleus, cytoplasm CXCR2 plasma membrane SMAD3 nucleus, cytoplasm GRB2 plasma membrane, SMAD4 nucleus, cytoplasm cytoplasm, nucleus TNFSF10 extracellular, plasma CXCR2 plasma membrane membrane LAT plasma membrane XIAP nucleus, cytoplasm CDK6 nucleus, cytoskeleton, cytosol ARFIP2 plasma membrane, cytosol, Golgi apparatus
TABLE-US-00003 TABLE 3 Detection of reference sets with SIMPL (b) Detection with SIMPL in bait-IN/prey-CIC format bait prey reference set PLU_mean PLU_sd SIMPL result DR1 DRAP1 3.969868 0.473734 positive GTF2F1 GTF2F2 3.240792 0.379569 positive LSM3 LSM2 3.034348 0.299742 positive CCND3 CDK6 2.724104 0.493514 positive RPA2 RPA3 2.722426 0.237577 positive CBLB GRB2 2.612294 0.620953 positive MAD2L1 MAD1L1 2.51995 0.268931 positive CASP2 CRADD 2.448596 1.268555 positive PEX14 PEX19 2.167057 0.005568 positive NCBP1 NCBP2 2.146787 0.111236 positive JUNB BATF 2.038777 0.198681 positive CDK2 CKS1B 1.882465 0.186788 positive LCP2 GRAP2 1.860639 0.213948 positive BAD BCL2L1 1.852165 0.24557 positive BAK1 BCL2L1 1.783491 0.097307 positive PEX19 PEX3 1.769572 0.165381 positive MAFG NFE2L1 1.717193 0.115782 positive PPP3CA PPP3R1 1.678497 0.161701 positive ATF3 DDIT3 1.637576 0.14194 positive GRB2 VAV1 1.388383 0.138295 positive HBA2 HBB 1.36129 0.270433 positive SKP1A BTRC 1.255416 0.139085 positive ERBB3 NRG1 1.219217 0.410585 positive LMNA RB1 1.183392 0.995994 positive PEX19 PEX11B 1.179537 0.206526 positive TNFSF10 TNFRSF10B 1.177707 0.062024 positive RIPK2 NOD1 1.173035 0.16047 positive PEX19 PEX16 1.091572 0.144247 positive RET NCBP2 1.060548 0.97354 positive HIF1A TP53 1.016036 0.093678 positive CD2 CD58 0.972098 0.086792 positive MAPK7 MAP2K5 0.948185 0.093095 positive IGF2 IGFBP4 0.943004 0.19349 positive MCM2 MCM3 0.938011 0.061255 positive PSMD4 RAD23A 0.91937 0.131715 positive RIPK2 PDGFRB 0.881284 0.139867 positive LMNA LMNB1 0.861232 0.12271 positive PSMD4 NOD1 0.845249 0.30456 positive NF2 HGS 0.835999 0.04363 positive S100A1 S100B 0.822118 0.046727 positive XIAP CASP9 0.810968 0.041818 positive LCP2 VAV1 0.78949 0.111015 positive CEBPG FOS 0.788308 0.340615 positive RCC1 RAN 0.747932 0.095913 positive GRB2 PTK2 0.740728 0.02354 positive CASP2 VAV1 0.69552 0.123926 positive RHOA ZBTB16 0.690993 0.13011 positive CDKN1B CCNA1 0.68555 0.01238 positive IFIT1 EIF3E 0.675818 0.297155 positive RAC1 ARFIP2 0.672191 0.186376 positive LCP2 NCK1 0.661524 0.061033 positive NR3C1 HSP90AA1 0.652489 0.163939 positive DDIT3 FOS 0.64182 0.090846 positive PDE4D RACK1 0.638768 0.074738 positive NR3C1 RELA 0.630063 0.089936 positive BDNF NTF4 0.628784 0.045742 positive RET FRS2 0.608223 0.111882 positive PTK2/FAK SRC 0.598863 0.074534 positive RAF1 RAP1A 0.577907 0.230264 positive BAK1 RAP1A 0.564385 0.033061 negative RIPK2 IGFBP4 0.555123 0.097446 negative HDAC1 RB1 0.539814 0.137497 negative PDGFRB PTPN11 0.53935 0.150515 negative IGF2 RPA3 0.524501 0.07459 negative CDKN1A CCNA1 0.516253 0.079258 negative BAD DCP1A 0.49879 0.053441 negative HDAC1 ZBTB16 0.491878 0.02719 negative ARF1 ARFIP2 0.491174 0.074615 negative AKT1 PDPK1 0.486209 0.068759 negative RHOA ARHGAP1 0.485965 0.023812 negative CEBPG CASP9 0.481506 0.108028 negative NF2 FOS 0.480537 0.188662 negative PTPN11 FRS2 0.474205 0.206277 negative MAFG TNFRSF10B 0.458591 0.030775 negative XIAP CASP7 0.451877 0.032686 negative XIAP ARFIP2 0.439407 0.055467 negative CGA CGB5 0.435412 0.070967 negative ORC2 MCM10 0.429797 0.002876 negative IGF2 HSP90AA1 0.403788 0.193402 negative SMAD3 SMAD4 0.398739 0.155898 negative SMAD4 DCP1A 0.398168 0.049793 negative B2M HLA-C 0.38573 0.035579 negative SKP1A SKP2 0.375745 0.047265 negative FGF1 NCK1 0.373869 0.057335 negative RHOA CCNA1 0.359663 0.295578 negative MCM2 MCM5 0.349833 0.016165 negative FEN1 PCNA 0.34944 0.020362 negative JUNB PTPN11 0.345289 0.003788 negative FEN1 GRAP2 0.341864 0.020429 negative MAFG PEX11B 0.341491 0.075287 negative FGF1 FGFR1 0.334291 0.103549 negative IFIT1 RELA 0.327506 0.089449 negative FGF1 RB1 0.324257 0.070172 negative HIF1A ARFIP2 0.32423 0.154696 negative TNFSF10 CDK6 0.316067 0.047023 negative XIAP CASP3 0.315704 0.101024 negative PEX14 NRG1 0.309753 0.021418 negative B2M HLA-B 0.305548 0.004724 negative ARF1 SKP2 0.30118 0.046346 negative CD2 GTF2F2 0.298631 0.033051 negative ORC2 ORC4 0.297486 0.067726 negative B2M HLA-A 0.288261 0.005242 negative ERBB3 DDIT3 0.279722 0.080593 negative CDK2 DDIT3 0.275186 0.028277 negative CBLB BCL2L1 0.272223 0.093711 negative LSM3 FGFR1 0.272014 0.024866 negative CXCL1 MAP2K5 0.269603 0.039369 negative IGF2 ARFIP2 0.265551 0.209521 negative GADD45A PDPK1 0.264868 0.032061 negative CRK PDGFRB 0.260567 0.012792 negative RAC1 BTRC 0.256375 0.024785 negative CD2 HGS 0.253155 0.023991 negative LGALS3 LGALS3BP 0.247573 0.036982 negative HDAC1 PPP3R1 0.247043 0.123296 negative PPP3CA FOS 0.245191 0.063541 negative MAPK7 PEX16 0.230529 0.116863 negative CDKN1B DRAP1 0.228777 0.013117 negative ORC2 HBB 0.227103 0.036994 negative LMNA PEX3 0.224467 0.085672 negative GRB2 LAT 0.222832 0.040559 negative HDAC1 LAT 0.221675 0.04251 negative BDNF MCM5 0.220349 0.037035 negative AKT1 PEX19 0.218458 0.169793 negative S100A6 S100B 0.217399 0.079923 negative NCBP1 S100B 0.216886 0.035444 negative SMAD4 LAT 0.214672 0.033353 negative GADD45A FRS2 0.210346 0.029615 negative GRAP2 LAT 0.194509 0.017824 negative TP53 UBE2I 0.194156 0.04363 negative LCP2 CRADD 0.193135 0.052165 negative BDNF MAD1L1 0.190383 0.031383 negative PRKAR2A SRC 0.18566 0.032192 negative NR3C1 RAD23A 0.18329 0.111034 negative SMAD1 SMAD4 0.181746 0.035855 negative CXCL1 CXCR2 0.177645 0.013534 negative FABP5 PTK2 0.173788 0.014644 negative HBA2 CKS1B 0.170859 0.01745 negative LGALS3 LSM2 0.162419 0.009571 negative S100A1 CASP3 0.15799 0.023719 negative PEX19 BATF 0.153584 0.025886 negative CRK UBE2I 0.152743 0.007107 negative SMAD1 GRB2 0.15124 0.022569 negative SMAD3 CXCR2 0.143507 0.00956 negative PDGFRB RAN 0.143002 0.019113 negative PDE4D EZR 0.136674 0.002281 negative ATF3 LMNB1 0.134442 0.017108 negative FABP5 S100A7 0.13371 0.037526 negative MAD2L1 NTF4 0.13325 0.019956 negative GTF2F1 NOD1 0.132609 0.005166 negative GRAP2 NFE2L1 0.131061 0.026992 negative CGA CD58 0.130693 0.00792 negative LSM3 LGALS3BP 0.12927 0.003416 negative RCC1 CASP7 0.124715 0.052199 negative PRKAR2A EZR 0.123433 0.038166 negative ORC2 UBE2I 0.118785 0.027584 negative CDKN1A PEX11B 0.113346 0.068613 negative AKT1 TCL1A 0.110536 0.013041 negative PSMD4 TCL1A 0.108356 0.009092 negative SKP1A CXCR2 0.106803 0.014761 negative S100A6 RAN 0.105488 0.018279 negative CXCL1 ORC4 0.10321 0.025572 negative MCM2 S100B 0.101573 0.012127 negative CXCL1 SMAD4 0.101152 0.019587 negative DDIT3 NRG1 0.096285 0.034975 negative MAD2L1 NFE2L1 0.081861 0.002436 negative ERBB3 PCNA 0.081193 0.027165 negative CGA MCM3 0.073756 0.018602 negative GADD45A PCNA 0.061656 0.002698 negative RPA2 S100A7 0.060628 0.003192 negative GRB2 PEX3 0.058757 0.026112 negative CEBPG ARHGAP1 0.058463 0.025787 negative DR1 NTF4 0.052933 0.007415 negative B2M PEX16 0.040836 0.010143 negative CGA NCBP2 0.036345 0.006647 negative B2M TP53 0.032948 0.007287 negative B2M RAD23A 0.015545 0.002994 negative
TABLE-US-00004 TABLE 4 Mitochondrial PPIs tested by SIMPL Bait Name Alias Transit signal Localization SDHA SDH1, SDHF N-terminal Matrix protein peripheral to inner membrane ETFA alpha-ETF N-terminal Matrix ETFA alpha-ETF N-terminal Matrix TIMM10 TIM10 middle Intermembrane space CHCHD6 MIC25 unknown Lipid-anchored to inner membrane in the intermembrane space. UQCRC2 QCR2, UQCR2 N-terminal Matrix side peripheral protein to inner membrane TIMM50 TIM50, TIM50L N-terminal Transmembrane protein at the inner mitochondrial membrane, exposing the C-terminus to the intermembrane space with interacting with TIMM23 N-terminal domain ATP5MC1 ATP5G1, ATP5A, ATP5G N-terminal Multipass transmembrane protein in the inner membrane with both N- and C-termini in the intermembrane space NDUFV1 UQOR1, CI51KD N-terminal Matrix protein peripheral to inner membrane PDHA1 PHE1A, PDHA, PDHAD N-terminal Matrix Prey Name Alias Transit signal Localization Function Reference SDHB SDH2 N terminal Matrix protein peripheral complex II Sun et al. Cell (2005) to inner membrane 121, 1043-1057 ETFB beta-ETF unknown Matrix ETF complex, Toogood et al, OXPHOS J Biol Chem (2004) complexes 279, 32904-32912 ETFRF1 LYRM5 unknown Matrix ETF complex, Floyd et al, Mol Cell OXPHOS (2016) 63, 621-632 complexes TIMM9 TIM9 middle Intermembrane space TIM9/10 complex. Webb et al, Mol Cell (2006) 21. 123-133 CHCHD3 MIC19 unknown Lipid-anchored to inner MICOS complex Kozjak-Pavlovic, membrane in the Cell Tissue intermembrane space. Res (2017) 367: 63-93 UQCRC2 QCR2, N-terminal Matrix side peripheral complex III Guo et al, Cell UQCR2 protein to inner membrane (2017) 170, 1247-1257 TIMM23 TIM23 unknown Inner membrane multiple TIM23 complex Geissler et al, Cell transmembrane protein (2002) 111, 507-518; with N-terminus in the Yamamoto et al, Cell intermembrane space 111 (2002), 519-528; Demishtein-Zohary et al, Cell Tissue Res (2017) 367, 33-41 ATP5MC1 ATP5G1, N-terminal Multipass transmembrane Fo complex of Rastogi et al, Nature ATP5A, protein in the inner ATPase (1999) 402. 263-268 ATP5G membrane with both N- and C-termini in the intermembrane space NDUFV3 CI9KD N-terminal Matrix protein peripheral N module of Guo et al, Cell (2017) to inner membrane complex I 170, 1247-1257 PDHB PHE1B, N-terminal Matrix Pyruvate Ciszak et al, PDHE1B, dehydrogenase J Biol Chem (2003) PDHBD complex 278, 21240-21246
TABLE-US-00005 TABLE 5 C. elegans references set studied by SIMPL Reference Bait Protein (IN-V5/HA) Prey Protein (IC/CIC-FLAG) Configuration ELISA Value Replicate 1 ELISA Value Replicate 2 # Set CDS Protein CDS Protein IN/IC IC/CIC IN/IC IC/CIC IN/IC IC/CIC PPI Description/Homologs Source Pubmed ID 1 Positive T05H4.2 FBXA-196 F46A9.4 SKR-2 Yes Yes 0.81 1.1 0.51 0.28 SKP1/F-Dox Domain CePRS 2 Positive Y45F10C.3 FBXA-215 F46A9.4 SKR-2 Yes Yes 0.84 0.73 0.58 0.42 SKP1/F-Box Domain CePRS 3 Positive ZK792.6 LET 60 AC7.2 SOC 2 Yes Yes 1.2 0.79 0.59 0.70 SHOC2/HRAS CePRS 4 Positive F53F10.5 NPP-11 K07F5.13 NPP-1 Yes inj. 1.8 — 0.92 — Nuclear pore complex components Nup54 LIT/EE 7531196 and Nup62 5 Positive F58F6.4 RFC-2 F44B9.8 F44B9.8 Yes inj. 0.66 — 0.88 — RFC5/RFC2 Replication factor subunits LIT/EE 15201901 6 Positive T10E9.1 T10E9.1 F46A9.4 SKR-2 Yes inj. 0.78 — 0.55 — SKP1/F-Box Domain CePRS 7 Positive T05G5.3 CDK-1 ZC168.4 CYB-1 inj. Yes — 1.1 — 0.80 Cyclin B2/Cyclin Dependent Kinase 1 LIT/EE 7575488 8 Positive C06G3.10 COGC-2 Y51H7C.6 COGC-4 n.c. n.c. — — — — COG2/COG4 (component of oligomeric LIT/EE 15047703 golgi complex 4) 9 Positive F10B5.6 EMB-27 B0511.9 CDC-26 inj. imj. — — — — Cdc26p and Cdc16p components of the LIT/EE 8895471 anaphase promoting complex 10 Positive K08B4.1 LAG-1 C32A3.1 SEL-8 n.c. n.c. — — — — CSL (CBF-1, Su(H), Lag-1)/Mastermind CePES 11 Positive F58A3.1 LDB-1 F46C8.5 CEH-14 n.c n.c — — — — LDB2 (LIM domain binding 2)/LHX3 CePRS (LIM homeobox 3) and LHX4 (LIM homeobox 4) 12 Positive M7.1 LET-70 F54G8.4 NHL-1 n.c n.c. — — — — UBE2D2 (ubiquitin conjugating enzyme CePRS E2 D2)/TRIM2 (tripartite motif containing 2) and TRIM3 (tripartite motif containing 3) 13 Positive F59E12.5 NPL-4.2 F19B6.2 UFD-1 n.c n.c. — — — — NPLOC4 (NPL4 homolog, ubiquitin LIT/EE 10811609 recognition factor)/UFD1 (ibiquitin recognition factor in ER associated degradation 1) 14 Positive F59A2.1 KPP-9 K01G5.4 RAN-1 n.c. n.c. — — — — RANBP1 (RAN binding protein 1)/RAN LIT/EE 7603572 (RAN, member RAS oncogene family) 15 Positive F21C3.4 RDE-2 F21C3.4 RDE-2 n.c. n.c. — — — — RDE2 RNA interference protein CePRS 16 Positive M03D4.1 ZEN-4 B0207.4 AIR-2 inj. inj. — — — — Aurora Kinase C/KIF23 CePRS 17 Positive Y37H2A.5 FBXA-210 F46S9.4 SKR-2 inj. inj. — — — — SKP1/F-Box Domain CePRS 18 Positive Y113G7B.5 FOG-2 T23G11.3 GLD-1 inj. inj. — — — — QKI (QKI, KH domain containing RNA CePRS binding)/Germline protein 19 Positive M7.1 LET-70 F16A11.1 F16A11.1 n.c. n.c. — — — — UBE2D2 (ubiquitin conjugating enzyme CePRS E2 D2)/RSPRY1 (ring finger and SPRY domain containing 1 20 Positive M7.1 LET-70 C45G7.4 C45G7.4 n.c n.c. — — — — UBE2D2 (ubiquitin conjugating enzyme CePRS E2 D2)/TRIM13 21 Positive ZK1098.8 MUT-7 F21C3.4 RDE-2 n.c. n,c. — — — — EXD3 (exonuclease 3′-5′ domain CePRS containing 3)/RDE2 involved in RNA interference 22 Positive R06F6.5 NPP-19 Y37E3.15 NPP-15 inj. inj. — — — — Nuclear pore complex components LIT/EE 16631361 Nup35 and Nup93 23 Positive Y49E10.14 PIE-1 F59B2.6 ZIF-1 inj. inj. — — — — mRNA 3′-UTR binding activity/ubiquitin- CePRS dependent protein 24 Positive T27F2.1 SKP-1 K08B4.1 LAG-1 n.c n.c. — — — — SNW1 (SNCS1 (CBF-1, Su(H), Lag-1)W CePRS domain containing 1)/ 25 Positive T07E3.4 T07E3.4 F46A9.4 SKR-2 inj. inj. — — — — SKP1/F-Box Domain CePRS 26 Positive Y39G10A.12 TPXL-1 K07C11.2 AIR-1 inj. inj. — — — — Interaction between Aurora A kinase LIT/EE 16054030 and microtubule binding protein TPX2 27 Positive Y54E5B.4 UBC-16 F54G8.4 NHL-1 n.c. n.c — — — — BBE2W (ubiquitin conjugating enzyme CePRS E2 W)/TRIM2 (tripartite motif containing 2) and TRIM3 (tripartite motif containing 3) Key to Abbreviations Yes = Line successfully established and analyzed by SIMPL ELISA platform n.c. = not cloned/cloning unsuccessful inj. = injected, but no transgenic line could be established with expression of both Bait and Prey CePRS = C. elegans Positive Reference Set LIT/EE = Literature derived and previously confirmed by yeast two-hybrid. Description/Homolog information from WormBase
TABLE-US-00006 SEQUENCE LISTING GP41-1 IN (WT) (SEQ ID NO: 1) CLDLKTQVQT PQGMKEISNI QVGDLVLSNT GYNEVLNVFP KSKKKSYKIT LEDGKEIICS EEHLFPTQTG EMNISGGLKE GMCLYVKE GP41-1 IC (WT) (SEQ ID NO: 2) MMLKKILKIE ELDERELIDI EVSGNHLFYA NDILTHN GP41-1 IN (C25) (SEQ ID NO: 3) CLDLKTQVQT PQGMKEISNI QVGDLVLSNT GYNEVLNVFP KSKKKSYKIT LEDGKEIICS EEHLFPTQTG EMNISGGLKE GMCLYVKEMM LKKILKIEEL GP41-1 IC (C25) (SEQ ID NO: 4) DERELIDIEV SGNHLFYAND ILTHN
REFERENCES
[0121] 1. Yao, Z., Petschnigg, J., Ketteler, R. & Stagljar, I. Application guide for omics approaches to cell signaling. Nat. Chem. Biol. 11, 387-397 (2015). [0122] 2. Petschnigg, J., Snider, J. & Stagljar, I. Interactive proteomics research technologies: recent applications and advances. Curr. Opin. Biotechnol. 22, 50-58 (2011). [0123] 3. Snider, J. et al. Fundamentals of protein interaction network mapping. Mol. Syst. Biol. 11, 848-848 (2015). [0124] 4. Suter, B., Kittanakom, S. & Stagljar, I. Two-hybrid technologies in proteomics research. Curr. Opin. Biotechnol. 19, 316-323 (2008). [0125] 5. Gogarten, J. P., Senejani, A. G., Zhaxybayeva, O., Olendzenski, L. & Hilario, E. Inteins: Structure, Function, and Evolution. Annu. Rev. Microbiol. 56, 263-287 (2002). [0126] 6. Shah, N. H. & Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446-461 (2014). [0127] 7. Aranko, A. S., Wlodawer, A. & Iwaï, H. Nature's recipe for splitting inteins. Protein Eng. Des. Sel. 27, 263-271 (2014). [0128] 8. Wood, D. W. & Camarero, J. A. Intein applications: From protein purification and labeling to metabolic control methods. J. Biol. Chem. 289, 14512-14519 (2014). [0129] 9. Dassa, B., London, N., Stoddard, B. L., Schueler-furman, O. & Pietrokovski, S. Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family. Nucleic Acids Res 37, 2560-2573 (2009). [0130] 10. Carvajal-Vallejos, P., Pallisse, Roser, Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686-28696 (2012). [0131] 11. Choi, J., Chen, J., Schreiber, S. L. & Clardy, J. Structure of the FKBP12-Rapamycin Complex Interacting with the Binding Domain of Human FRAP. Science 273, 239-242 (1996). [0132] 12. Aranko, a S. et al. Structure-based engineering and comparison of novel split inteins for protein ligation. Mol. Biosyst. 10, 1023-34 (2014). [0133] 13. Machleidt, T. et al. NanoBRET-A Novel BRET Platform for the Analysis of Protein-Protein Interactions. ACS Chem. Biol. 10, 1797-1804 (2015). [0134] 14. Venkatesan, K. et al. An empirical framework for binary interactome mapping. Nat. Methods 6, 83-90 (2009). [0135] 15. Lievens, S. et al. Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor. Mol. Cell. Proteomics 13, 3332-3342 (2014). [0136] 16. Trepte, P. et al. LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells. Mol. Syst. Biol. 14, e8071 (2018). [0137] 17. Braun, P. et al. An experimentally derived confidence score for binary protein-protein interactions. Nat. Methods 6, 91-97 (2009). [0138] 18. Lemmon, M. a. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117-1134 (2010). [0139] 19. Zheng, Y. et al. Temporal regulation of EGF signalling networks by the scaffold protein Shc1. Nature 499, 166-71 (2013). [0140] 20. Pylayeva-Gupta, Y., Grabocka, E. & Bar-Sagi, D. RAS oncogenes: Weaving a tumorigenic web. Nat. Rev. Cancer 11, 761-774 (2011). [0141] 21. Petschnigg, J. et al. The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Nat. Methods 11, 585-92 (2014). [0142] 22. Yao, Z. et al. Resource A Global Analysis of the Receptor Tyrosine Kinase-Resource A Global Analysis of the Receptor. Mol. Cell 65, 347-360 (2017). [0143] 23. Feig, L. A. & Cooper, G. M. Inhibition of NIH 3T3 Cell Proliferation by a Mutant ras Protein with Preferential Affinity for GDP. Mol. Cell. Biol. 8, 3235-3243 (1988). [0144] 24. Waas, W. F. & Dalby, K. N. Transient Protein-Protein Interactions and a Random-ordered Kinetic Mechanism for the Phosphorylation of a Transcription Factor by Extracellular-regulated Protein Kinase 2. J. Biol. Chem. 277, 12532-12540 (2002). [0145] 25. Garai, A. et al. Specificity of Linear Motifs That Bind to a Common Mitogen-Activated Protein Kinase Docking Groove. Sci. Signal. 5, ra74 (2012). [0146] 26. Floyd, B. J. et al. Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function Article Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function. Mol. Cell 63, 621-632 (2016). [0147] 27. Jackson, T. D., Palmer, C. S. & Stojanovski, D. Mitochondrial diseases caused by dysfunctional mitochondrial protein import. Biochem. Soc. Trans. 46, 1225-1238 (2018). [0148] 28. Kozjak-Pavlovic, V. The MICOS complex of human mitochondria. Cell Tissue Res. 367, 83-93 (2017). [0149] 29. Ciszak, E. M., Korotchkina, L. G., Dominiak, P. M., Sidhu, S. & Patel, M. S. Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase. 278, 21240-21246 (2003). [0150] 30. Yaish, P., Gazit, A., Gilon, C. & Levitzki, A. Blocking of EGF-Dependent Cell Proliferation by EGF Receptor Kinase Inhibitors. Science 242, 933-935 (1988). [0151] 31. Roberts, A. W., Stilgenbauer, S., Seymour, J. F. & Huang, D. C. S. Venetoclax in Patients with Previously Treated Chronic Lymphocytic Leukemia. Clin. Cancer Res. 23, 4527-4534 (2017). [0152] 32. Souers, A. J. et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 19, 202-208 (2013). [0153] 33. Stagljar, I. The power of OMICs. Biochem. Biophys. Res. Commun. 479, 607-609 (2016). [0154] 34. Barrios-Rodiles, M. et al. High-Throughput Mapping of a Dynamic Signaling Network in Mammalian Cells. Science 307, 1621-1625 (2005). [0155] 35. Lemmens, I. et al. Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells. Nucleic Acids Res. 31, e75 (2003). [0156] 36. Fields, S. & Song, O. A Novel Genetic System to Detect Protein-Protein Interactions. Nature 340, 245-246 (1989). [0157] 37. Galarneau, A., Primeau, M., Trudeau, L. & Michnick, S. W. β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein-protein interactions. Nat. Biotechnol. 20, 619-622 (2002). [0158] 38. Ramachandra, N. et al. Self-Assembling Protein Microarrays. Science 305, 86-91 (2004). [0159] 39. Olhovsky, M. et al. OpenFreezer: a reagent information management software system. Nat. Methods 8, 612-613 (2011). [0160] 40. Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301 (1995). [0161] 41. Kotlyar, M., Pastrello, C., Malik, Z. & Jurisica, I. IID 2018 update: Context-specific physical protein-protein interactions in human, model organisms and domesticated species. Nucleic Acids Res. 47, D581-D589 (2019). [0162] 42. Kotlyar, M. et al. In silico prediction of physical protein interactions and characterization of interactome orphans. Nat. Methods 12, 79-84 (2014). [0163] 43. Boxem, M. et al. A Protein Domain-Based Interactome Network for C. elegans Early Embryogenesis. Cell 134, 534-545 (2008). [0164] 44. Simonis, N. et al. Empirically controlled mapping of the Caenorhabditis elegans protein-protein interactome network. Nat. Methods 6, 47-54 (2009).
[0165] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0166] It should be understood that the materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
[0167] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0168] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0169] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
[0170] Other embodiments are set forth within the following claims.