Methods of enzyme purification using g-proteins as controllable affinity probes

Abstract

A method is provided for the use of G-proteins as affinity probes for the isolation of their endogenous binding partners. In one embodiment, the method uses Rheb or a Rag heterodimer to isolate soluble endogenous mTOR complex 1 (mTORC1).

Description

PATENT METHODS OF ENZYME PURIFICATION USING G-PROTEINS AS CONTROLLABLE AFFINITY PROBES BACKGROUND

[0001] Large and small G-proteins, also known as GTP-binding proteins or GTPases, are an abundant family of enzymes that respond to and regulate various cellular processes including neurotransmission, growth factor signaling, organelle function, and cell growth. Small GTPases can have different binding properties depending upon whether they are GTP- or GDP-nucleotide bound. Throughout these different nucleotide-state changes, context-dependent binding partners are recruited and released, promoting cellular activities for various signaling pathways.

[0002] The phosphatidylinositol 3-kinase-related kinase (PIKK) family of signaling proteins has been implicated in conditions ranging from cancer and metabolic diseases to aging and growth malformations. The PIKK family is composed of a group of evolutionarily related kinases, namely mechanistic target of rapamycin (mTOR), ataxia- telangiectasia mutated (ATM), ataxia- and Rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PK), suppressor of morphogenesis in genitalia (SMG1), and transformation / transcription domain-associated protein (TRAPP). Members of the PIKK family form known stable, soluble complexes with other proteins, which have functionally distinct activities, mechanisms of regulation, and signaling outputs.

[0003] The mechanistic target of rapamycin (mTOR) protein is a large kinase that acts as the catalytic subunit of two well-defined functionally independent complexes, mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). mTORC1 is a central regulator of mammalian cell growth that is dysregulated in a number of human diseases, including metabolic syndromes, aging and cancer. Therefore, mTORC-specific inhibitors are of interest for clinical treatment of mTOR complex-driven diseases. The use of recombinant enzymes as targets for drug development can be complicated by protein misfolding, incomplete expression, an overloaded expression system, and cellular toxicity. Moreover, modifications that facilitate isolation, purification, and / or detection of the enzymes, such as tags or labels, can alter the endogenous state or function of the protein. In contrast, endogenous enzymes are in their physiological form, at natural equilibrium and with normal interactions and activation states.

[0004] Endogenous enzymes have classically been purified through various multi- step chromatography methods. However, no such protocols have been developed forPATENT mTORC1, likely due to the size and instability of the multi-protein complex, which can cause components to dissociate during the extensive / rigorous separation process. To date, mTORC1 purification has typically relied on expression of tagged components of the complex, such as tags on Raptor, in cultured eukaryotic cells, which allows for rapid purification that does not require chromatography. mTOR has also been purified by antibody pulldown, which creates an immune complex used for rapid non- chromatography based purification, or by exploiting the rapamycin-mTOR interaction. All of these processes introduce confounding variables into subsequent assessment of the enzyme, and require high costs to generate sufficient yields for use in applications such as high-throughput drug screening for identification of potential enzyme-targeting therapeutics. In addition to being expensive and inefficient, these methods do not necessarily isolate endogenous mTORC1, hampering their overall utility in research.

[0005] A cost-effective method that overcomes the limitations of tags or undissociated-supplemental components, such as immunocomplexes, is needed to provide large quantities of soluble, endogenous enzymes and enzyme complexes, such as mTORC1. SUMMARY OF THE INVENTION

[0006] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and any and all such combinations of embodiments are intended to fall within the scope of the present invention.

[0007] The invention provides methods of isolating endogenous mTORC1 from various cellular sources using isolated recombinant small-GTPases as controllable affinity probes, or bait, to isolate interacting proteins, or targets, from other sources. In an exemplary embodiment, recombinant RagA and RagC are expressed in bacteria and used to isolate endogenous mTORC1 from mammalian cells. Given that Rag GTPase binding to Raptor is nucleotide dependent, the elution of mTORC1 from recombinant Rags is readily achieved by nucleotide-switching. This can be achieved by chelation of the Mg2+divalent ion that is required for the stabilization of both the RagA-GTP and RagC-GDP nucleotide binding and their interaction with Raptor, mediated by its switch-I domainPATENT (Wang et al.2000; Zhang et al.2000; Wittinghofer et al.2011; Rudack et al.2012). Addition of recombinant GATOR1, a known GAP for RagA / B (Bar-Peled et al.2013), could be an alternative elution approach.

[0008] Accordingly, GTPases can be used in a controlled manner for purification of interacting proteins in their endogenous states. This represents an advantage over traditional chromatography approaches, immuno-purification approaches where an immunocomplex remains bound to the protein, or tag-based purification approaches where a defined protein state is not necessarily isolated and either the tag or its residual sequence remains. Moreover, the use of an isolated small-G protein for affinity purification allows for large quantities of interacting proteins to be purified from abundant sources at a fraction of the cost of other purification methods.

[0009] In one embodiment, the invention provides an in vitro method for isolating a binding partner of a GTPase, the method comprising: (a) contacting (i) an active form of a GTPase with (ii) a sample comprising the binding partner, wherein the binding partner binds to the GTPase to form a GTPase-binding partner complex; (b) capturing the GTPase-binding partner complex; and (c) eluting the binding partner from the GTPase; thereby isolating the binding partner. In a particular embodiment, the binding partner is mechanistic target of rapamycin complex 1 (mTORC1) and wherein the GTPase is a Rag heterodimer. In an additional embodiment, the binding partner is mTORC1α and the GTPase is Rheb. In another embodiment, the binding partner is mechanistic target of rapamycin complex 2 (mTORC2) and wherein the GTPase is selected from Rap1 and RasC.

[0010] A further embodiment of the invention is an in vitro method for isolating a mechanistic target of rapamycin complex 1 (mTORC1), the method comprising: (a) contacting (i) an active Rag heterodimer comprising an affinity tag with (ii) a sample comprising mTORC1, wherein the mTORC1 binds to the Rag heterodimer to form a Rag- mTORC1 complex; (b) capturing the Rag-mTORC1 complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rag heterodimer; and (c) eluting the mTORC1 from the Rag heterodimer; thereby isolating mTORC1. The method can optionally further comprise (d) contacting the mTORC1 with an active Rheb protein comprising an affinity tag, wherein the Rheb protein binds to mTORC1α to form a Rheb-mTORC1α complex; (e) capturing the Rheb-mTORC1α complex on an affinity substrate, wherein the affinity substrate comprises a bindingPATENT partner of the affinity tag on the Rheb protein; and (f) eluting the mTORC1α from the Rheb heterodimer. Alternatively or in addition, the method can optionally further comprise analyzing the mTORC1 or the mTORC1α to identify one or more components of the complex.

[0011] An additional embodiment is an in vitro method for separating mTORC1α from mTORC1β, the method comprising: (a) contacting (i) an active Rheb protein comprising an affinity tag with (ii) a sample comprising mTORC1α and mTORC1β, wherein the mTORC1α binds to the Rheb protein to form a Rheb-mTORC1α complex; (b) capturing the Rheb-mTORC1α complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rheb protein; (c) optionally, collecting eluent comprising the mTORC1β; and (d) eluting the mTORC1α from the Rheb protein; thereby separating mTORC1α from mTORC1β.

[0012] Also provided is a method for producing a preparation of isolated GTP-bound RagA protein or RagB protein, the method comprising: (a) transforming a cell with a nucleic acid comprising a sequence encoding a RagA protein or a RagB protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the cell expresses a GST-tagged RagA protein or a GST-tagged RagB protein; (b) isolating the GST-tagged RagA or GST-tagged RagB protein by affinity purification with a glutathione-conjugated affinity substrate; (c) releasing nucleotide from the GST-tagged RagA or GST-tagged RagB protein; and (d) incubating the GST-tagged RagA or GST-tagged RagB protein with (i) GTP, GTPgS, or GDPnHP and (ii) Mg2+; thereby producing a preparation of isolated GTP-bound RagA protein or RagB protein.

[0013] An additional embodiment is a method for producing a preparation of isolated GDP-bound RagC protein or RagD protein, the method comprising: (a) transforming a cell with a nucleic acid comprising a sequence encoding a RagC protein or a RagD protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the cell expresses a GST-tagged RagC protein or a GST-tagged RagD protein; (b) isolating the GST-tagged RagC or GST-tagged RagD protein by affinity purification with a glutathione-conjugated affinity substrate; (c) releasing nucleotide from the GST-tagged RagC or GST-tagged RagD protein; and (d) incubating the GST-tagged RagC or GST- tagged RagD protein with GDP and Mg2+; thereby producing a preparation of isolated GDP-bound RagC protein or RagD protein. In some embodiments, the transformed cell is a bacterial cell.PATENT

[0014] In another embodiment, the invention provides a method for preparing an active Rag heterodimer, the method comprising: (a) producing a preparation of isolated GTP-bound RagA protein or RagB protein, comprising: (i) transforming a first cell with a nucleic acid comprising a sequence encoding a RagA protein or a RagB protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the first cell expresses a GST-tagged RagA protein or a GST-tagged RagB protein; (ii) isolating the GST-tagged RagA or GST-tagged RagB protein by affinity purification with a glutathione-conjugated affinity substrate; (iii) releasing nucleotide from the GST-tagged RagA or GST-tagged RagB protein; and (iv) incubating the GST-tagged RagA or GST- tagged RagB protein with (i) GTP, GTPgS, or GDPnHP and (ii) Mg2+; thereby producing a preparation of isolated GTP-bound RagA protein or RagB protein; (b) producing a preparation of isolated GDP-bound RagC protein or RagD protein, comprising: (i) transforming a second cell with a nucleic acid comprising a sequence encoding a RagC protein or a RagD protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the second cell expresses a GST-tagged RagC protein or a GST-tagged RagD protein; (ii) isolating the GST-tagged RagC or GST- tagged RagD protein by affinity purification with a glutathione-conjugated affinity substrate; (iii) releasing nucleotide from the GST-tagged RagC or GST-tagged RagD protein; and (iv) incubating the GST-tagged RagC or GST-tagged RagD protein with (i) GTP, GTPgS, GDPnHP, or GDP and (ii) Mg2+; thereby producing a preparation of isolated, homogenous-nucleotide-bound RagC protein or RagD protein; and (c) combining the preparation of step a) with the preparation of step b); thereby preparing an active Rag heterodimer. In some embodiments, one or both of the first cell and the second cell is a bacterial cell.

[0015] A further aspect of the invention is a method for screening a candidate molecule for its ability to compete with a GTPase for binding to a binding partner of the GTPase, the method comprising: (a) contacting (i) an active form of a GTPase with (ii) a sample comprising the binding partner, wherein the binding partner binds to the GTPase to form a GTPase-binding partner complex; (b) capturing the GTPase-binding partner complex; (c) contacting the GTPase-binding partner complex with the candidate molecule; and (d) determining whether the binding partner is released from the GTPase in the presence of the candidate molecule; whereby if the binding partner is released from the GTPase, the candidate molecule is able to compete with the GTPase for binding to thePATENT binding partner. In certain embodiments, the GTPase is selected from a Rag heterodimer and Rheb.

[0016] Another embodiment is a method for screening a candidate molecule for its ability to compete with an active Rag heterodimer for binding to mTORC1, the method comprising: (a) contacting (i) the Rag heterodimer with (ii) a sample comprising mTORC1, wherein mTORC1 binds to the Rag heterodimer to form a Rag-mTORC1 complex; (b) capturing the Rag-mTORC1 complex; (c) contacting the Rag-mTORC1 complex with the candidate molecule; and (d) determining whether mTORC1 is released from the Rag heterodimer in the presence of the candidate molecule; whereby if mTORC1 is released from the Rag heterodimer, the candidate molecule is able to compete with the Rag heterodimer for binding to mTORC1.

[0017] Also provided is a kit comprising: (i) a composition comprising an active Rag heterodimer, wherein at least one monomer of the heterodimer comprises an affinity tag; and (ii) an affinity substrate comprising a binding partner of the affinity tag. In one embodiment, the affinity tag is GST and the binding partner is glutathione. In one embodiment, the affinity substrate is agarose beads. In a particular embodiment, the Rag heterodimer is bound to the affinity substrate via the affinity tag. In some embodiments, the kit further comprises an elution buffer, wherein the elution buffer comprises a magnesium chelating agent and / or a monovalent chloride salt. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG.1A-1F show a schematic diagram of one embodiment of a method of the invention. (1A) GST-tagged RagA and RagC are expressed and purified. (1B) RagA is switched to a GTP-bound state and RagC is switched to a GDP-bound state. (1C) RagA- GTPgS and RagC-GDP are combined to form a RagA-GTPgS / RagC-GDP dimer. (1D) RagA-GTPgS / RagC-GDP dimers are incubated with cell lysate containing mTORC1. (1E) Lysate is flowed onto glutathione beads, which bind the GST tag on the RagA- GTPgS / RagC-GDP dimer. Dimer-bound mTORC1 is captured on the beads and separated from other lysate proteins. (1F) mTORC1 is eluted by displacement of GTPgS / GDP.

[0019] FIG.2A-2F show that conserved interacting regions of Raptor and Rag GTPases highlight the utility of the affinity assay in all mTOR homolog-expressing species except plants. (2A) The crystal structure of human Raptor with the dimer of human RagA-GTP with RagC-GDP at 3.18 Ångström with the interacting regionsPATENT between the super-complex. The structure was obtained from PDB file 6U62 (7). A conserved protein sequence analysis comparing human versus all homolog-containing species for RagA and RagC (2B) or Raptor (2C-2E) is superimposed onto PDB 6U62 using ConSurf software. Conserved protein sequences for human Raptor versus Raptor homologs from all species (2C), all animals (2D), and plants (2E) are shown. (2F) Raptor conserved protein sequences for Arabidopsis thaliana (plant) versus homologs from all other plants superimposed onto Arabidopsis thaliana Raptor PDB 5WBI. Computed conservation scores are assigned color grades 1-9, going from variable to conserved, as indicated.

[0020] FIG.3A-3B show that in silico-modified mTORC1 structures representing the mTORα and mTORβ isoforms exhibit shared Rag-binding to mTORC1 isoforms, and distinct Rheb binding to mTORC1β isoform, with likely monomer formation of the mTORC1β. The atomic-structure of RagA / C bound to mTORC1 (pdb 6sb2) was used for in-silico modification. The full-length human atomic-structure of Rheb, predicted by AlphaFold 2 (afRHEB) was matched to the partial atomic structure of Rheb found on mTORC1 (pdb 6bcu). (3A) After matching mTORC1-Rheb (pdb 6bcu) and afRHEB to mTORC1-RagA / C (pdb 6sb2), afRHEB was kept on mTORC1-RagA / C and is presented. (3B) Amino-acids 1-1600 of mTOR were deleted from the mTORC1 dimer structure to form mTORC1β. Full length mTOR found in (pdb 6bcu and 6sb2) is mTORC1α.

[0021] FIG.4A-4I show a schematic diagram of one embodiment of a method of the invention. (4A) RagA-GTP / RagC-GDP are incubated with a lysate containing the various mTORC1 forms that contain mTORα and mTORβ isoforms and other lysate proteins. (4B) RagA-GTP / RagC-GDP captures both mTORα and mTORβ mTORC1 isoforms via to binding to Raptor. (4C) mTORC1 isoforms are eluted off of RagA / RagC by chelating magnesium. (4D) Rheb is expressed and purified from bacteria. (4E) Rheb is switch to a GTP-state. (4F) Rheb is incubated with the eluted mTORC1 isoforms, or lysates. (4G) Rheb captures mTORα isoform mTORC1 due to the presence of the mTOR binding site only in full-length mTOR. (4H) mTORβ isoform mTORC1 is collected from the supernatant. (4I) mTORα isoform mTORC1 is eluted off of Rheb by chelating magnesium.

[0022] FIG.5A-5B show Coomassie stain (5A) and SDS-page Western immunoblot (5B) of 8% acrylamide gel of recombinant human GST-RagA and GST-RagC expressedPATENT in E. coli and purified on glutathione beads. Experiments were repeated at least three time.

[0023] FIG.6A-6B show (6A) Coomassie stain (upper panel) and immunoblotting (lower panel) of Rag-isolated mTORC1 from MEF lysates. (6B) Rag-isolated mTORC1 from MEFs were immunoblotted against the indicated proteins. Experiments were repeated at least twice. mTORC1 was isolated by Rag GST-pulldown, lysed and boiled.

[0024] FIG.7A-7B show Coomassie stain (7A) and SDS-page Western immunoblot (7B) of 6% acrylamide gel of eluate. GST-tagged RagA-GTPgS / RagC-GDP dimers were incubated with mTORC-1-containing lysate from HEK293 cells. mTORC1 was eluted from glutathione beads with NaCl+EDTA. Beads were washed with HEPES buffer and GST-bound proteins were eluted with glutathione. mTORC1 components mTOR, Raptor, and mLST8 were detected in first eluate; GST-tagged RagA and RagC were detected in second eluate.

[0025] FIG.8 shows Rag-isolation from HEK293A cells. Affinity-isolated samples were subjected to immunoblotting against the indicated proteins. The cropped boxes show the input with corresponding affinity-isolated samples, loaded at different sides of the same SDS-PAGE, processed together, and shown at the same exposure, brightness, and contrast. The experiment was repeated at least three times.

[0026] FIG.9 shows protein aggregation-based thermal denaturation of mTORC1, measured by fluorescence quenching in a thermal cycler quantitative PCR. Large protein complexes exhibit high-fluorescence at lower temperatures. Binding of Torin1 or rapamycin to mTORC1 shifted temperature-induced protein aggregation by up to 7°C and 2°C, respectively. Results are the average of three independent experimental repeats, each with four, three, and three technical repeats, respectively.

[0027] FIG.10A-10B show activity of the eluted mTORC1 in a high-throughput kinase assay. The ability of eluted mTORC1 to phosphorylate its downstream target, 4EBP1 was measured by dot blot (10A) using an anti-pT37 / 464EBP1 primary antibody and horseradish peroxidase-conjugated secondary antibody. Signal intensity versus mTORC1 concentration is shown in (10B).

[0028] FIG.11A-11B show Rag-isolation of mTORC1 from bovine liver tissue. Lysates of affinity-isolated mTORC1 were subjected to immunoblotting against the endogenous indicated proteins (11A) or were incubated with ATP and 4E-BP1 in an in vitro kinase assay (11B). The ability of eluted human mTORC1 to phosphorylate itsPATENT downstream target 4E-BP1 at T37 / 46 was measured by dot blot analysis (11B). mTORα was detected with polyclonal mTOR antibody ab2732 (Abcam), and mTORβ with rabbit monoclonal antibody 2983 (Cell Signaling Technologies). Experiments were carried out on three individual liver samples. In 11A, samples containing input and affinity-isolated proteins were processed on separate SDS-PAGE gels.

[0029] FIG.12 shows that Rheb-GTP and combined RagA-GTP / RagC-GDP can isolate mTORC1. However, Rheb-GTP can only isolate mTORα-isoform-containing mTORC1, whereas RagA-GTP / RagC-GDP can isolate mTORα and mTORβ forms of mTORC1 in cell-types that contain both isoforms of mTOR.

[0030] FIG.13 shows a GST-pulldown assay of isolated and eluted mTORC1 using the RagA-GTP / RagC-GDP method. DETAILED DESCRIPTION OF THE INVENTION

[0031] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

[0032] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, 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 is related.

[0033] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0034] All of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.PATENT I. Definitions

[0035] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0036] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0037] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0038] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are included.

[0039] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

[0040] The terms “isolated” or “purified” are used interchangeably and refer to material that is substantially free from components that normally accompany the material as it is found in its endogenous or native state.

[0041] A “polynucleotide,” as used herein can include one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or theirPATENT analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0042] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g., for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Unless specifically indicated, peptides are indicated with the N-terminus of the left and the sequence is written from the N-terminus to the C-terminus.

[0043] The term “binding” refers to the interaction between a corresponding pair of molecules that exhibit mutual affinity, typically specific or non-specific binding or interaction, including biochemical, physiological, and / or pharmaceutical interactions. Biological binding defines a type of interaction that occurs between pairs of molecules including proteins, nucleic acids, glycoproteins, carbohydrates, hormones and the like. The binding can result from hydrogen bonding, hydrophobic forces, van der Waals forces, or ionic bonding, for example.

[0044] “Binding partner” refers to a molecule that can undergo binding with a particular target. Examples of binding partners include antibody / antigen, antibody / hapten, enzyme / substrate, enzyme / inhibitor, enzyme / cofactor, binding protein / substrate, carrier protein / substrate, lectin / carbohydrate, receptor / ligand, monomers that form a dimer, complementary strands of nucleic acid, protein / nucleic acid repressor / inducer, and virus / ligand.

[0045] “Binding affinity” generally refers to the strength of the sum total of non- covalent interactions between a single binding site of a molecule (e.g., a GTPase) and its binding partner (e.g., an enzyme). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., GTPase and enzyme) in their endogenous forms. For example, in some instances, the GTPase is a multimeric complex, such as a heterodimer. In some instances, the enzyme is a multimeric complex. In such instances where one orPATENT both of the binding partners occur endogenously as complexes, the 1:1 interaction refers to an interaction between the endogenous form of each binding partner.

[0046] The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art. Low-affinity binding partners generally bind slowly and tend to dissociate readily, whereas high-affinity binding partners generally bind faster and tend to remain bound longer.

[0047] The affinity or avidity of a molecule for its target can be determined experimentally using any suitable method known in the art, e.g., flow cytometry, enzyme- linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA® or BIACORE™ analysis). Direct binding assays as well as competitive binding assay formats can be readily employed. (See, e.g., Berzofsky et al., “Antibody- Antigen Interactions,” In Fundamental Immunology, Paul, W. E., ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: New York, N.Y. (1992).) The measured affinity of a particular interaction between binding partners can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other binding parameters (e.g., KDor Kd, Kon, Koff) are made with standardized solutions of binding partners, and a standardized buffer, as known in the art.

[0048] The term “elution” refers to methods that cause dissociation of two or more molecules that have affinity to one-another. Elution can be achieved, for example, by competition of the binding molecules with a molecule having affinity for the same site; by altered ionic interactions of the two molecules by changing the ionic strength, pH, and / or temperature of the solution; by removal of components that maintain the molecular interaction (e.g., ions in the GTPase), or by modification of allosteric interactions that indirectly affect the affinity of the molecules for each other. The elution strategy can be directed to either the target molecule / complex and its components (e.g., mTOR, Raptor, mLST8), or to the bait molecule / complex and its components (e.g., GTPase, nucleotide, ion).

[0049] A “label” is a detectable compound that can be conjugated directly or indirectly to a molecule, so as to generate a “labeled” molecule. The label can be detectable on its own (e.g., radioisotope labels or fluorescent labels), or can be indirectly detected, for example, by catalyzing chemical alteration of a substrate compound orPATENT composition that is detectable (e.g., an enzymatic label) or by other means of indirect detection (e.g., biotinylation).

[0050] A molecule in its “active form” is able to carry out its chemical or biological function. Examples of such functions include binding to a binding partner, catalyzing a reaction, and activating a signaling cascade. In the context of the present invention, a GTPase is in its active form when coordinated with a divalent ion, preferably magnesium.

[0051] An “active agent” is an ingredient that is intended to furnish biological activity. The active agent can be in association with one or more other ingredients. An “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.

[0052] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An “inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule.

[0053] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[0054] One such non-limiting example of a sequence alignment algorithm is described in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly availablePATENT software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)), can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used. Other resources for calculating identity include methods described in Computational Molecular Biology (Lesk ed., 1988); Biocomputing: Informatics and Genome Projects (Smith ed., 1993); Computer Analysis of Sequence Data, Part 1 (Griffin and Griffin eds., 1994); Sequence Analysis in Molecular Biology (G. von Heinje, 1987); Sequence Analysis Primer (Gribskov et al. eds., 1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988). II. GTP-Binding Proteins

[0055] As used herein, the terms GTP-binding protein, G-protein, and GTPase are used interchangeably and refer to polypeptides that can bind to and hydrolyze the nucleotide guanosine triphosphate (GTP) to guanosine diphosphate (GDP). GTPases can also bind non-hydrolyzable analogs of GTP or GDP. The GTPases can be naturally occurring molecules, subunits of naturally occurring molecules, or artificially engineered molecules. GTPases are typically active in their GTP-bound state and inactive in their GDP-bound state.

[0056] Because nucleotide binding affinity for GTPases is high, the GTPase- nucleotide state in cells is regulated by interacting proteins, such as GTPase activating proteins (GAP), which promote the GTPase to hydrolyze GTP to GDP + Pi (inorganicPATENT phosphate) (Barbacid 1987). GDP nucleotide exchange factors (GEF) then bind the GDP-bound GTPase and promote GDP unloading, which allows for new GTP reloading (Vetter et al.2001). The changes in GTP or GDP-state are associated with large molecular rearrangements in the protein structure at switch-I and switch-II domains (also known as G2 and G3 domains), which interact with the gamma-phosphate of GTP. In the GDP state, these untethered interactions release the switch-domains away from the nucleotide. Moreover, these rearrangements can affect other structural regions within the GTPase such as the interswitch region that when affected can in some GTPases drive distant N-terminal structural changes. These altered molecular states promote the binding and release of target proteins. Therefore, nucleotide-cycling in GTPases functions as a regulated switch-mechanism controlling interacting protein-binding and signal transduction.

[0057] Nucleotide binding affinity and GTP hydrolysis are key controlled steps in GTPase nucleotide-cycling that use conserved domains to form a common active-site found within most GTPases. The mechanisms of nucleotide coordination within the active-site have been well characterized at the biochemical and atomic-structural levels. In particular, common to most small GTPases is the G1 domain amino acid sequence GxxxxGKS / T (RAS-family GTPases), which serves as part of the p-loop that coordinates and neutralizes the gamma-phosphate of GTP. The G4 domain sequence N / TKxD coordinates the nucleotide guanosine-ring, together with the G5 domain-arginine coordinating an oxygen on the guanosine-ring, which serve to specify the type of nucleotide accepted. A review of the GTP binding motif is provided by Kjeldgaard et al. (1996).

[0058] In the GTP-bound state a divalent ion, typically a magnesium ion (Mg2+), coordinates with the gamma and beta phosphates of GTP or beta-alpha phosphates of GDP, with the conserved serine in the G1 domain, and with the threonine in the Switch I domain, to form a complete active site. The magnesium ion is essential for maintaining affinity to nucleotide; the enzymes exhibit up to 1000-fold lower affinity to nucleotide if the magnesium is absent (Zhang et al.2000). Moreover, the GTPase activity is magnesium ion dependent. In addition, an Arginine “finger” in GAP proteins stabilizes the gamma-phosphate and a glutamine in the Switch II domain to coordinate the hydrolysis in the active site of the GTPase. Taken together, the structural state is controlled by nucleotide and Mg2+ ion forming the basis for a G-protein’s binding statePATENT and catalytic activity. Non-hydrolyzable analogs of GTP maintain the same structural interactions with the enzyme and Mg2+, but do not hydrolyze to produce GDP or Pi, and do not release the enzyme from its nucleotide-bound state (e.g. GTP-bound).

[0059] A GTPase that is “GTP-bound” includes one that is bound to GTP or to non- hydrolyzable GTP analogs, such as GTPgS [(2S,3R,4S,5S)-5-(2-amino-6-oxo-3H-purin- 9-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydroxyphosphinothioyl hydrogen phosphate and GDPnHP [[(2R,3S,4R,5R)-5-(2-Amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxyoxolan- 2-yl]methoxy-oxidophosphoryl]oxy-(phosphonatoamino)phosphinate tetralithium.

[0060] GTPases for use in the invention include any polypeptide comprising domains that can be coordinated to form an active site as described above and having nucleotide- binding and hydrolytic activity. Nucleotides such as GTP, GTPgS, GDPnHP, and GDP, include their deoxy forms.

[0061] Examples of GTPases suitable for use in the invention include members of the Ras superfamily, including members of its multiple subfamilies, such as the Arf, Miro, Rab, Ran, Rap, Ras, RGK, Rheb, Rho, and Rit subfamilies.

[0062] In some embodiments, the GTPase is a monomer. In some embodiments, the GTPase is a multimer, such as a dimer. In a particular embodiment, the GTPase is a heterodimer. For example, the GTPase can be a Rag heterodimer, as described below. GTPase Interactors

[0063] In nature, G-protein association and release is spatially localized within cellular compartments, is tightly regulated, and can occur only in cells where the GTPase and its binding partner co-exist. Subcellular localization of GTPases can include the cytosol, subplasmalemmal actin mesh, cholesterol-rich microdomains, nucleus, and endomembranes, for example, the plasma membrane, the nuclear membrane, Golgi, and endosomes, including multivesicular bodies. GTPase binding partners include proteins and protein complexes, peptide oligomers, phospholipids, gangliosides, nucleosides, nucleic acids, glycosaminoglycans, and small organic and inorganic compounds. mTOR Complexes

[0064] mTORC1 is a GTPase-interacting complex that contains the mTOR, Raptor, mLST8, proline-rich AKT substrate 40 kDa (PRAS40), DEP-domain-containing protein (Deptor) proteins; mTORC2 contains mTOR, mLST8, Deptor, rapamycin-insensitive companion of mTOR (Rictor), protein observed with Rictor-1 (Protor-1), and mammalianPATENT stress-activated protein kinase interacting protein (mSIN1). The complexes can also include non-protein components, such as ATP, and molecules that bind to components of the complex, for example, rapamycin.

[0065] mTORC1 is regulated by signals controlling nutrient availability, such as amino acids, glucose, and growth factors. One of the best-characterized downstream substrates of mTORC1 is the translation initiation inhibitor 4EBP1, whose phosphorylation by mTORC1 promotes protein synthesis. As such, mTORC1 is the target of the drugs rapamycin and Torin1, which bind to and inhibit the activity of mTORC1 via different sites and mechanisms within the kinase.

[0066] Many of the signals activating mTORC1 are transduced via the small GTPase Rheb, which associates with and activates mTORC1 directly on the surface of the lysosome (Avruch et al.2009). As a part of an amino-acid sensing signal transduction mechanism, mTORC1 is localized to the lysosome through binding and recruitment by the Rag family of small GTPases. Rags directly interact with Raptor in mTORC1, forming a super-complex, which recruits mTORC1 to the lysosomal surface via Rag- dependent interactions with lysosomal surface proteins such as Ragulator and others (Sancak et al.2008; Sancak et al.2010).

[0067] In particular, RagA or RagB forms a heterodimer with RagC or RagD, which binds to, recruits, and releases mTORC1. The nucleotide state of each Rag, dictates whether interactions with mTORC1 are favored or dis-favored (Shen et al.2017). For example, a heterodimer of GTP-bound RagA or RagB and GDP-bound RagC or RagD favors binding of to mTORC1 and leads to its lysosomal recruitment and activation. Hydrolysis of GTP or facilitated nucleotide exchange results in a nucleotide state switch wherein RagA or RagB becomes inverted (GDP-bound), and RagC or RagD becomes inverted (GTP-bound). This nucleotide switch destabilizes the Rag heterodimer- mTORC1 interaction, and results in the separation of the super-complex and the release of mTORC1.

[0068] Switch-region or hydrolytic-site mutant forms of RagA, RagB, RagC, RagD, and Rheb can abolish nucleotide switching or hydrolysis, and result in a locked GDP- or GTP-bound state, respectively (Sanack et al.2008; Lee et al.2018). Examples of locked GTPase suitable for use in the invention include Q66L RagA-GTP; T21L RagA-GDP; Q99L RagB-GTP; T54L RagB-GDP; Q120L RagC-GTP; S75L RagC-GDP; Q121L RagD-GTP; S77L RagD-GDP; Q64L Rheb1-GTP; and D60I Rheb1-GDP. Accordingly,PATENT permanent nucleotide-state GTPases, such as mutants or non-hydrolyzable nucleotide- bound GTPases, constitutively maintain or lose interactions with their cognate GTPase interactors. III. Methods of Isolation and Screening

[0069] The present invention relies on the use of small G-proteins as affinity probes to isolate soluble GTPase binding partners in their endogenous form. Mammalian RagA and RagC bind the mTORC1-component Raptor with high affinity when RagA is bound to GTP and RagC to GDP (Kim et al.2008; Sancak et al.2008; Sancak et al.2010). To build a cost-effective tool that isolates endogenous mTORC1, one embodiment of the invention comprises the strategy shown in FIG.1A-1F. The basis of this strategy is to express GST-tagged recombinant human RagA and RagC in Escherichia coli (E. coli) (FIG.1A), set these GTPases into their high-mTORC1-affinity nucleotide state: GST- RagA-GTP and GST-RagC-GDP (FIG.1B), and utilize the GTPase heterodimer as an affinity probe to isolate mTORC1 from various cellular sources by GST affinity purification using glutathione beads (FIG.1C-1F).

[0070] By replacing GTP with the slow / non-hydrolysable GTPγS, the potential hydrolysis of GST-RagA-GTP by intrinsic GTPase activity or GTPase Activating Proteins (GAPs) present in lysates is suppressed (FIG.1B). At the end, mTORC1 can be eluted by changing the GST-Rag GTPase nucleotide-state by simple chelation of magnesium which disrupts nucleotide binding within the Rags (Wittinghofer et al.2011; FIG.1F).

[0071] In one aspect, the invention provides an in vitro method for isolating a binding partner of a GTPase, the method comprising: (a) contacting (i) an active form of a GTPase with (ii) a sample comprising the binding partner, wherein the binding partner binds to the GTPase to form a GTPase-binding partner complex; (b) capturing the GTPase-binding partner complex; and (c) eluting the binding partner from the GTPase. The method can be applied to any known GTPase / binding partner pair. The method can also be applied to a sample to identify unknown GTPase binding partners. In one embodiment, the binding partner is mTORC1 and the GTPase is selected from a Rag heterodimer and Rheb. In one embodiment, the binding partner is mTORC2 and wherein the GTPase is selected from Rap1 and RasC.PATENT

[0072] A particular embodiment provides an in vitro method for isolating mTORC1 comprising (a) contacting (i) an active Rag heterodimer comprising an affinity tag with (ii) a sample comprising mTORC1, wherein the mTORC1 binds to the Rag heterodimer to form a Rag-mTORC1 super-complex; (b) capturing the Rag-mTORC1 super-complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rag heterodimer; and (c) eluting the mTORC1 from the Rag heterodimer.

[0073] Atomic-scale in silico modelling of the Rag-Raptor binding interface across the animal and plant kingdoms indicated that this strategy potentially can isolate mTORC1 from various animal cells, but not plant cells, since they lack Rag homologs and the Raptor binding interface is not conserved between humans and plants (Tatebe et al.2017; FIG.2A-2F).

[0074] Some cells and tissues express distinct isoforms of mTOR: mTORα and mTORβ, which are capable of forming a complex with Raptor and mLST8. This additional level of complexity can result in mixed mTORC1 isolates, depending on the source of lysate. As used herein, “mTORC1α” refers to an mTORC1 comprising the mTORα isoform, and “mTORC1β” refers to an mTORC1 comprising the mTORβ isoform. If not otherwise specified, mTORC1 can comprise the α or β isoform of mTOR.

[0075] In silico-modified mTORC1 structures of mTORC1α and mTORC1β show that RagA / C binding to mTOR is shared between mTORC1α and β isoforms, whereas the Rheb binding site is deleted from mTORC1β. Therefore, Rheb can only bind the mTORC1α isoform (FIG.3A-3B).

[0076] Accordingly, one embodiment provides an in vitro method for isolating mTORC1α comprising (a) contacting (i) an active Rheb protein comprising an affinity tag with (ii) a sample comprising mTORC1α, wherein the mTORC1α binds to Rheb to form a Rheb-mTORC1α complex; (b) capturing the Rheb-mTORC1α complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rheb protein; and (c) eluting the mTORC1α from the Rheb protein.

[0077] Active Rheb protein can also be used in a method for separating mTORC1 isoforms. Accordingly, in one aspect, the invention provides an in vitro method for separating mTORC1α from mTORC1β, the method comprising: (a) contacting (i) an active Rheb protein comprising an affinity tag with (ii) a sample comprising mTORC1α and mTORC1β, wherein the mTORC1α binds to Rheb to form a Rheb-mTORC1αPATENT complex; (b) capturing the Rheb-mTORC1α complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rheb protein; (c) collecting eluent comprising the mTORC1β; and (d) eluting the mTORC1α from the Rheb protein; thereby separating mTORC1α from mTORC1β.

[0078] Methods of the invention can be combined with one another in any manner. For example, an in vitro method for isolating mTORC1 or for purifying mTORC1α or for separating mTORC1α from mTORC1β can comprise a method using Rag heterodimers to isolate TORC1 from a lysate and a method using Rheb to separate mTORC1α from mTORC1β. One such combination method is show in FIG.4A-4I.

[0079] The above methods can also identify molecules that bind to mTORC1. Accordingly, the invention provides a method of screening for an mTORC1 binding partner, by isolating mTORC1 or an isoform thereof, as described herein, and analyzing members of the complex.

[0080] Samples subjected to methods of the invention can be from lysates of cells, tissues, or organs or from blood. The sample can be from any organism or cell type that comprises a GTPase binding partner or an mTORC1 or mTORC2 binding partner. Cells, tissues, or organs can be isolated from an organism or cultured in vitro. In one embodiment, the sample is a cell lysate, preferably a mammalian cell lysate. Preferably, the sample is not from a plant.

[0081] The “active Rag heterodimer” is a heterodimer comprising a RagA-GTP or RagB-GTP subunit and a RagC-GDP or RagD-GDP subunit. In specific embodiments, the active Rag heterodimer is selected from the group consisting of RagA-GTP / RagC- GDP; RagA-GTP / RagD-GDP; RagB-GTP / RagC-GDP; and RagB-GTP / RagD-GDP.

[0082] An “active Rheb protein” is Ras homolog enriched in brain (Rheb) in its GTP- bound form.

[0083] GTPase binding partner complexes can be captured via any matrix / substrate interaction. Preferably, the GTPase comprises an affinity tag and is captured on an affinity substrate. As used herein the term “affinity substrate” refers to a matrix or support comprising an affinity tag. An affinity substrate can include, for example, a resin, a bead, a particle, a membrane, and a gel. In a preferred embodiment, the affinity substrate is an agarose bead.

[0084] A variety of affinity tags are known in the art. Non-limiting examples include albumin-binding protein; alkaline phosphatase; biotin; calmodulin-binding peptide;PATENT chloramphenicol acetyl transferase; cellulose-binding domain; chitin-binding domain; choline-binding domain; dihydrofolate reductase; epitopes such as AU1, AU5, E2, FLAG, HSV, KT3, Myc, and T7; galactose-binding protein; glutathione S-transferase; Glu-Glu (EE); hemagglutinin; histidine affinity tag; LacZ; maltose-binding protein; poly- amino acids, such aspolyarginine, polyaspartate, polycysteine, polyhistidine, and polyphenylalanine; Protein A; Protein C; Protein G; S-tag and S1-tag; streptavidin; streptavidin-binding protein; thioredoxin; TrpE; and VSV-G. In one embodiment, the affinity tag is GST and the affinity substrate comprises glutathione.

[0085] The binding partner can be eluted from the GTPase using ionic elements, such as salts or chelators. Elution can also be achieved using small molecules or peptides that affect the GTPase (such as GEF protein or peptides, or peptides that act as a GAP), that affect the binding interface of the GTPase and binding partner, or that target the GTPase- binding partner interaction and cause dissociation. Alternatively, elution can be promoted by addition of nucleotide. In certain embodiments, the binding partner, such as mTORC1, is eluted with a monovalent chloride salt, a Mg2+ chelating agent, or both. Examples of monovalent chloride salts include NaCl and KCl. Examples of Mg2+ chelating agents include EDTA and EGTA.

[0086] After elution, the binding partner can optionally be incubated with Mg2+ and further purified and / or concentrated by known methods, including dialysis and filtration.

[0087] Methods for producing recombinant Rag proteins are also provided. One embodiment is a method for producing a preparation of isolated GTP-bound RagA protein or RagB protein, the method comprising: (a) transforming a cell with a nucleic acid comprising a sequence encoding a RagA protein or a RagB protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the cell expresses a GST-tagged RagA protein or a GST-tagged RagB protein; (b) isolating the GST-tagged RagA or GST-tagged RagB protein by affinity purification with a glutathione-conjugated affinity substrate; (c) releasing nucleotide from the GST-tagged RagA or GST-tagged RagB protein; and (d) incubating the GST-tagged RagA or GST-tagged RagB protein with GTP or a non-hydrolyzable analogue thereof, such as GTPgS or GDPnHP, and Mg2+.

[0088] Another embodiment is a method for producing a preparation of isolated GDP-bound RagC protein or RagD protein, the method comprising: (a) transforming a cell with a nucleic acid comprising a sequence encoding a RagC protein or a RagDPATENT protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the cell expresses a GST-tagged RagC protein or a GST-tagged RagD protein; (b) isolating the GST-tagged RagC or GST-tagged RagD protein by affinity purification with a glutathione-conjugated affinity substrate; (c) releasing nucleotide from the GST-tagged RagC or GST-tagged RagD protein; and (d) incubating the GST-tagged RagC or GST- tagged RagD protein with GDP and Mg2+.

[0089] A further embodiment is a method for preparing an active Rag heterodimer, the method comprising: (a) producing a preparation of isolated GTP-bound RagA protein or RagB protein, comprising: (i) transforming a first cell with a nucleic acid comprising a sequence encoding a RagA protein or a RagB protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the first cell expresses a GST-tagged RagA protein or a GST-tagged RagB protein; (ii) isolating the GST-tagged RagA or GST-tagged RagB protein by affinity purification with a glutathione-conjugated affinity substrate; (iii) releasing nucleotide from the GST-tagged RagA or GST-tagged RagB protein; and (iv) incubating the GST-tagged RagA or GST-tagged RagB protein with GTP or a non-hydrolyzable analogue thereof, such as GTPgS or GDPnHP, and Mg2+; (b) producing a preparation of isolated GDP-bound RagC protein or RagD protein, comprising: (i) transforming a second cell with a nucleic acid comprising a sequence encoding a RagC protein or a RagD protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the second cell expresses a GST-tagged RagC protein or a GST-tagged RagD protein; (ii) isolating the GST-tagged RagC or GST- tagged RagD protein by affinity purification with a glutathione-conjugated affinity substrate; (iii) releasing nucleotide from the GST-tagged RagC or GST-tagged RagD protein; and (iv) incubating the GST-tagged RagC or GST-tagged RagD protein with GTP, a non-hydrolyzable analogue thereof, such as GTPgS or GDPnHP, or GDP, and Mg2+; and (c) combining the preparation of step (a) with the preparation of step (b).

[0090] In one embodiment, the Rag protein or the Rheb protein is released from the affinity substrate using, for example, glutathione or reduced glutathione.

[0091] In a preferred embodiment, Rag proteins are expressed in a bacterial cell. The advantage of expressing Rag proteins in prokaryotic cells is that their endogenous binding partners are not present, so their purification is easier and the yields are higher. In addition, expressing the Rag monomers in separate populations of cells permits the expressed Rag proteins to be switched to an all GTP-bound state (for RagA and RagB) orPATENT an all GDP-bound state (for RagC or RagD), resulting in higher yields of active heterodimers. Accordingly, methods in which Rag proteins are expressed in eukaryotic cells can comprise an optional elution step prior to affinity purification.

[0092] In one embodiment, Rheb protein is expressed in a bacterial cell.

[0093] In one embodiment, the GTPase is bound to non-hydrolyzable GTP, for example, GTPgS or GDPbHP. An advantage to switching to the GTP-bound state using non-hydrolyzable analogs of a nucleotide is that the active state of the GTPase can be maintained under conditions of exposure to endogenous GAP proteins. In particular, a sample for use in methods of the invention may contain GAP proteins that could induce GTPase-mediated hydrolysis of GTP to GDP + Pi, which would disrupt the intended active state of the GTPase.

[0094] A further aspect of the invention involves methods of screening candidate molecules to identify molecules that can interrupt the GTPase-binding partner interaction. In one embodiment, a method is provided for screening a candidate molecule for its ability to compete with a GTPase for binding to a binding partner of the GTPase, the method comprising: (a) contacting (i) an active form of a GTPase with (ii) a sample comprising the binding partner, wherein the binding partner binds to the GTPase to form a GTPase-binding partner complex; (b) capturing the GTPase-binding partner complex; (c) contacting the GTPase-binding partner complex with the candidate molecule; and (d) determining whether the binding partner is released from the GTPase in the presence of the candidate molecule; whereby if the binding partner is released from the GTPase, the candidate molecule is able to compete with the GTPase for binding to the binding partner.

[0095] A particular embodiment is a method for screening a candidate molecule for its ability to compete with an active Rag heterodimer for binding to mTORC1, the method comprising: (a) contacting (i) the Rag heterodimer with (ii) a sample comprising mTORC1, wherein mTORC1 binds to the Rag heterodimer to form a Rag-mTORC1 complex; (b) capturing the Rag-mTORC1 complex; (c) contacting the Rag-mTORC1 complex with the candidate molecule; and (d) determining whether mTORC1 is released from the Rag heterodimer in the presence of the candidate molecule; whereby if mTORC1 is released from the Rag heterodimer, the candidate molecule is able to compete with the Rag heterodimer for binding to mTORC1.

[0096] Another embodiment is a method for screening a candidate molecule for its ability to compete with Rheb for binding to mTORC1, the method comprising: (a)PATENT contacting (i) an active Rheb protein with (ii) a sample comprising mTORC1, wherein mTORC1 binds to the Rheb protein to form a Rheb-mTORC1 complex; (b) capturing the Rheb-mTORC1 complex; (c) contacting the Rheb-mTORC1 complex with the candidate molecule; and (d) determining whether mTORC1 is released from the Rheb protein in the presence of the candidate molecule; whereby if mTORC1 is released from the Rheb protein, the candidate molecule is able to compete with Rheb for binding to mTORC1. IV. Kits

[0097] The invention provides a kit comprising: (i) a composition comprising an active Rag heterodimer, wherein at least one monomer of the heterodimer comprises an affinity tag; and (ii) an affinity substrate comprising a binding partner of the affinity tag. In one embodiment, the affinity tag is GST and the binding partner is glutathione. In one embodiment, the affinity substrate is agarose beads. The kit can comprise the Rag heterodimer and the affinity substrate separately, or the affinity substrate can be provided with the Rag heterodimer bound. In some embodiments, the kit further comprises an elution buffer. The elution buffer can comprise, for example, one or more ion chelating agents and / or a monovalent chloride salt.

[0098] The invention also provides a kit comprising: (i) a composition comprising an active Rheb protein comprising an affinity tag; and (ii) an affinity substrate comprising a binding partner of the affinity tag. In one embodiment, the affinity tag is GST and the binding partner is glutathione. In one embodiment, the affinity substrate is agarose beads. The kit can comprise the active Rheb protein and the affinity substrate separately, or the affinity substrate can be provided with the Rheb protein bound. In some embodiments, the kit further comprises an elution buffer. The elution buffer can comprise, for example, one or more ion chelating agents and / or a monovalent chloride salt. EXAMPLES

[0099] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.PATENT Example 1. Preparation of RagA-GTP and RagC-GDP Rag GTPase Expression in Bacterial Cells

[0100] Full-length human RagA and RagC were cloned in-frame with N-terminally tagged GST in pGEX4t.1 vector system, where translation is controlled by the Lac operon. pGEX plasmids were transformed in BL21 DE3 competent E. coli cells; positive clones were grown under ampicillin selection. BL21 competent pGEX-RagA and pGEX- RagC, were grown in 250L LB media each at 37°C degrees overnight. Cultures were then inoculated into 0.5-1L LB for protein expression. Upon reaching 0.6 optical density, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 400 μM, and incubated at 37°C for 4-5 hours prior to bacteria pelleting by centrifugation. Cell pellets were resuspended in HEPES buffer (40 mM HEPES, 150mM NaCl, 5mM MgCl2and cOmplete™ protease inhibitor cocktail from Roche) and sonicated with a probe-sonicator. Protein lysates were incubated with Glutathione-Beads (Pierce Glutathione Agarose, Thermo Fisher Scientific) at 4°C for 1-2 hours, followed by bead isolation by centrifugation and 10 times wash with HEPES buffer.

[0101] Gel electrophoresis of purified samples, followed by staining with Coomassie Brilliant Blue G-250 (Bio-Rad) for 1-12 hours and washing in deionized water, demonstrated that the primary bands in isolated GST-RagA and GST-RagC corresponded to the expressed proteins, a 55 and 70 kDa protein, respectively (FIG.5A-5B). Additional bands suggested that partial proteolysis / degradation of the Rags occurred in the bacterial lysates, which was minimal, indicating that human RagA and RagC can be readily produced in vitro. The GST-RagA isolate also contained a larger unknown protein (~65 kDa). Purified Rag GTP / GDP State Switching and Resetting

[0102] To set RagA and RagC in their mTORC1 high-affinity states, two different methods were utilized. In the first method, cleared bacterial lysate was incubated with glutathione-conjugated agarose beads and washed 10 times with HEPES buffer. GST- RagA and GST-RagC were isolated from glutathione-conjugated beads with 10mM reduced glutathione. Rag proteins were processed independently unless otherwise indicated.

[0103] To release the nucleotides from the Rag proteins, purified RagA or RagC was placed in 10 kDa dialysis tubing and incubated with 10 mM EDTA 1 hour at roomPATENT temperature. RagA or RagC dialysis tubing was placed in HEPES dialysis buffer without MgCl2for 24 hours at 4°C, renewing buffer 2-3 times. While still in the dialysis tubing, purified RagA was incubated with 1mM GTPγS and mixed; purified RagC was incubated with 1mM GDP and mixed. Purified RagA or RagC was incubated with 10 mM MgCl2for 30 minutes at room temperature. Dialysis tubing was then placed in dialysis buffer HEPES with MgCl2for 24-48 hours at 4°C, renewing dialysis buffer 2-3 times.

[0104] Purified RagA or RagC was concentrated using a 10 kDa Centricon® filter, and protein concentration was quantified by nano-drop 280nm wavelength measurement. Equal molar concentrations of purified, nucleotide-switched RagA and RagC were combined to make RagA-GTPγS / RagC-GDP dimers.

[0105] In a second method, glutathione-beads containing GST-proteins were switched to a no-MgCl2-Wash-Buffer (40mM HEPES, 150mM NaCl), and incubated at room temperature with 10 mM EDTA for 1 hour on rotation to remove magnesium from the catalytic core of the GTPases and thereby dissociate bound nucleotides. EDTA was removed by washing the samples 10 times with MgCl2-Wash-Buffer (40mM HEPES, 150mM NaCl, 5mM MgCl2). Samples were incubated with the specified nucleotides for 1 hour at room temperature, on rotation, and washed 5-10 times with MgCl2-Wash- Buffer. Example 2. mTORC1 Purification from Mammalian Cell Lysates Using RagA- GTPγS / RagC-GDP

[0106] To prepare samples from which to extract mTORC1, HEK293A cells and mouse embryonic fibroblasts (MEFs) were grown in 2D culture with DMEM high glucose (Thermo Fisher Scientific) and 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific).0.5g-1g of each liver was homogenized by mincing the tissue with razor blades in the appropriate amount of ice-cold Lysis buffer (40mM HEPES, 150mM NaCl, 5mM MgCl2, 0.2% CHAPS with cOmplete protease inhibitor cocktail from Roche). Mammalian cells were lysed in ice-cold Lysis buffer and passed through a 23G needle 10 times. Samples were centrifuged at max-speed for >30 minutes at 4°C. Lysates were either directly incubated with nucleotide-switched GST-Rag GTPases or were dialyzed using 100 kDa dialysis tubing (Spectrum labs) for 24 hours in Lysis buffer, renewing buffer 2-3 times.

[0107] Equal molar concentrations of purified, nucleotide-switched RagA and RagC were combined to make RagA-GTPgS / RagC-GDP dimers. In particular, glutathionePATENT beads containing nucleotide-switched GST-Rag GTPases were combined at a 1:1 ratio and incubated with lysates for 2-4 hours on rotation at 4°C. Samples were washed 10 times with Lysis-buffer without protease inhibitors, and proteins were recovered by boiling the samples for 5-10 minutes in Laemmli buffer with 5% 2-mercaptoethanol, or by elution.

[0108] To release mTORC1 components from RagA-GTPgS / RagC-GDP by elution, samples were washed 5x with Target-No-MgCl2-Wash-buffer (40mM HEPES, 150mM NaCl, 0.2% CHAPS), then incubated with Target-No-MgCl2-EDTA-Wash-buffer (40mM HEPES, 500mM NaCl, 0.2% CHAPS, 5mM EDTA) for 3 hours at room temperature. Supernatant / eluate was collected and 10mM MgCl2was added and incubated at 4C° for 1 hour. High NaCl containing eluate was diluted with Target-No-NaCl-Wash-buffer (40mM HEPES, 0.2% CHAPS, 5mM MgCl2) to reach a final NaCl concentration of 150mM. The Target-No-MgCl2-Wash-buffer and Target-No-MgCl2-EDTA-Wash-buffer eluates were each concentrated using a 100kDa cutoff Amicon Concentrator. Glutathione-beads containing GST-Rag GTPases, GST-4E-BP1 or GST-Rheb were incubated with 10mM Reduced-Glutathione (Thermo Fisher Scientific) in MgCl2-Wash- Buffer (40mM HEPES, 150mM NaCl, 5mM MgCl2). Eluate was concentrated using a 10kDa cutoff Amicon Concentrator.

[0109] Distinct proteins of the expected molecular weights were eluted from cell lysates. Coomassie staining of samples from MEF lysates revealed a band at 250 kDa, which corresponded to mTOR, as assessed by immunoblotting (FIG.6A). In particular, an SDS-page gel that had been stained with Coomassie blue was transferred to a nitrocellulose membrane so that the Coomassie stain was detectible on the membrane. After immunoblotting against mTOR, both the anti-mTOR HRP signal and the Coomassie staining could be detected simultaneously on the Amersham Imager 680 (GE Healthcare). Furthermore, the Rag-affinity pulldown elute was enriched in mTOR, Raptor and LST8, in contrast to the mTORC2’s Rictor, as judged by immunoblotting analysis (FIG.6B). Likewise, affinity pulldown and elution of HEK293A cell lysate samples readily isolated mTORC1-sized component proteins, which were confirmed to be Raptor, mTOR and LST8 by immunoblotting (FIG.7A-7B). Moreover, the affinity pulldown ‘wash’ condition lacked mTORC1, suggesting specificity to the chelation- mediated elution step (FIG.7B). Furthermore, immunoblotting only detected minor levels of mTORC2 components Rictor and Sin1 (FIG.8).PATENT Example 3. mTOR Thermal Dissociation Assay

[0110] Rapamycin and Torin1 bind to mTORC1 at different sites and inhibit its activity via different mechanisms. To determine whether purified mTORC1 maintained its native structural state, eluted endogenous soluble mTORC1 was incubated with 50 nM rapamycin or 250 nM Torin1 for 30 minutes prior to addition of 1:1000 SYPRO® Orange. Thermal dissociation of mTORC1 from rapamycin or Torin1 was measured by real-time melting curve detection using quantitative PCR.

[0111] Structural changes in the protein complex are detected by changes in the interaction with the hydrophobic dye, thus providing a measure of the stability or dissociation of the protein-protein interactions upon treatment. Results are shown in FIG. 9. Both rapamycin and Torin1, two specific mTORC1 inhibitors, affected denaturation / aggregation, demonstrating that mTORC1 was present in the eluate and retained sufficient structure to bind rapamycin and Torin1. Example 4. mTORC1 Kinase Assay

[0112] The activity of purified mTORC1 was assessed by its ability to phosphorylate its downstream target, 4E-BP1, in the presence of the small GTPase Rheb, which associates with and activates mTORC1. Rheb and 4E-BP1, each with an N-terminal GST-tag, were expressed separately in competent E. coli and purified as described in Example 1 for RagA and RagC. Purified Rheb was loaded with either GDP or GTPγS; GTP-bound Rheb is known to activate mTORC1 (Garami et al.2003).

[0113] Increasing concentrations of HEK293A-isolated mTORC1 were incubated with a set amount of GST-4E-BP1 in 40mM HEPES buffer with 5mM MgCl2, and GST- Rheb in either the GTP-bound or GDP-bound nucleotide state for 30 min at room temperature. 1mM ATP was added to each mixture and incubated for 1hr at 37°C. Similarly, bovine isolate mTORC1 was incubated with a set amount of GST-4E-BP1 in 40mM HEPES buffer with 5mM MgCl2. Control samples were incubated with water; treated samples were incubated with 1mM ATP for 1hr at 37°C.

[0114] Kinase reaction was terminated by boiling for 5-10 minutes in Laemmli sample buffer with 5-10% 2-mercaptoethanol. 25–50 μg of lysate was resolved by SDS- PAGE and transferred to 0.2 μm nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked with EveryBlot Blocking Buffer (Bio-Rad Laboratories) for 1 hour. For dot-blots, 2.5 μl of kinase reaction assays were dotted onto nitrocellulose membranes (Bio-Rad Laboratories), dried, then blocked with 2.5% non-fat milk.PATENT

[0115] Primary antibodies were diluted in blocking buffer and incubated over-night at 4°C. Incubations in horseradish peroxidase (HRP)-conjugated secondary antibody (GE healthcare) were performed in blocking buffer at room temperature for 1 hour. Membranes were captured with an Amersham Imager 680 (GE Healthcare), using SuperSignal™ West Pico PLUS Chemiluminescent Substrate or SuperSignal™ West Femto Maximum Sensitivity Substrate from Thermo Scientific.

[0116] A significant increase in the phosphorylation of 4E-BP1 T37 / 46 was observed when Rheb-GTPγS was added to the reaction mixtures containing increasing amounts of isolated mTORC1, compared to the addition of Rheb-GDP (FIG.10A-10B). 4E-BP1 was phosphorylated by both conditions at the highest mTORC1 amount, indicating that the eluted kinase is constitutively active. Taken together, these data demonstrate that mTORC1 can be eluted from the Rag GTPases ex vivo, and that eluted mTORC1 is catalytically functional, thus retaining the structural features necessary for its further activation by Rheb. Example 5. mTORC1 Isolation and Purification from Tissue

[0117] To test whether the present methods could be scaled beyond cultured cells and applied to animal tissues, an abundant source of mTORC1, the assay was performed on lysates of bovine liver. In murine and human liver, two isoforms of mTOR exist: the full- length form designated as mTORα, and a splice-isoform known as mTORβ (Panasyuk et al., 2009). Tagged-protein overexpression studies using HEK293 cells have demonstrated that both mTORα and mTORβ can form mTORC1 in cells, however, this has never been demonstrated in vivo, nor has the presence of mTORC1-β been demonstrated physiologically.

[0118] Immunoblotting for mTORC1 components in purified mTORC1 from bovine liver confirmed that the affinity pulldown isolated Raptor at 150 kDa, LST8 at 35 kDa, mTORα at ~280 kDa, and a ~80 kDa mTOR-sized protein (FIG.11A) that was strongly immunoreactive to anti-mTOR antibody and arguably corresponds to the mTORβ- isoform. Rabbit monoclonal mTOR (Cell Signaling Technologies #2983) strongly detected mTORβ. A polyclonal antibody against mTOR (ab2732, Abcam) was utilized for the detection of full-length mTORα in these samples in line with a previous study (Dong et al.2022).

[0119] Interestingly, an in vitro kinase assay of the isolate indicated that bovine mTORC1 was catalytically active as it phosphorylated 4E-BP1 at T37 / 46 (FIG.11B).PATENT Collectively, these data indicate that the Rag affinity-assay can be used to scale up mTORC1 isolation, and that mTORα and mTORβ may form distinct mTOR complexes that coexist in the same tissue. Example 6. GST-Rheb, GST Expression and Nucleotide Loading

[0120] Rheb protein with an N-terminal GST-tag (pGEX_4t.1 vector) and GST alone were each expressed separately in BL21 competent E. coli. Expression was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) for 5 hours and cells were lysed by sonication in HEPES buffer. Cell debris was separated from the soluble fraction of lysate by high-speed centrifugation. Cleared lysate was incubated with glutathione-conjugated agarose beads and washed 10 times with HEPES buffer. GST-Rheb and GST were isolated from glutathione-conjugated beads with 10mM reduced glutathione.

[0121] To release the nucleotides from the Rheb, purified Rheb was placed in 10 kDa dialysis tubing and incubated with 10 mM EDTA and 10 mM EGTA for 1 hour at room temperature in an empty beaker (without dialysis buffer). Activated charcoal can be used for chelation in combination with EDTA / EGTA.

[0122] Rheb dialysis tubing was placed in HEPES dialysis buffer without MgCl2for 24 hours at 4°C, renewing buffer 2-3 times. Purified Rheb was incubated with 1mM GTPgS and mixed. Purified Rheb was incubated with 10 mM MgCl2for 30 minutes at room temperature in an empty beaker (without dialysis buffer). Dialysis tubing was then placed in dialysis buffer HEPES with MgCl2for 24-48 hours at 4°C, renewing dialysis buffer 2-3 times.

[0123] Purified Rheb and GST proteins were concentrated using a 10 kDa Centricon® filter, mixed with glycerol to a final concentration of >50% glycerol, and stored at -80°C. Alternatively, purified Rheb and GST proteins were directly mixed with glycerol to a final concentration >50% Glycerol and stored at -80°C. Example 7. Purification of mTORC1 from Cell Lysate Using Rheb-GTPγS and RagA- GTPγS / RagC-GDP

[0124] To prepare samples from which to extract mTORC1, HEPG2 cells were lysed in HEPES buffer containing 0.2% CHAPS with 10% glycerol. Lysate was dialyzed using 100 kDa dialysis tubing for 24 hours in HEPES buffer containing 0.2% CHAPS at 4°C, renewing buffer 2-3 times. Alternatively, lysate can be used directly, without dialysis.

[0125] GST-tagged Rheb-GTP or GST-tagged RagA-GTPgS and GST-tagged RagC- GDP were incubated with the lysate for 4 hours at 4°C. The lysate mixtures were flowedPATENT onto glutathione-conjugated agarose beads, which were then washed 10 times with HEPES buffer.

[0126] Gel electrophoresis demonstrated that distinct proteins of the expected molecular weights were isolated (FIG.12). mTORα, mTORβ, and mLST8 were detected by Western immunoblotting. Detected proteins show that the Rheb-isolate of mTORC1 is highly enriched for mTORα whereas the RagA / RagC-isolate contains a mixture of mTORα and mTORβ forms of mTORC1. Example 8. Purification of mTORC1 from mTORC1 Eluate Using Rheb-GTPγS

[0127] To prepare samples from which to extract mTORC1, mTORC1 protein was prepared as described above. GST-tagged Rheb-GTP or a GST control were incubated with the isolated mTORC1 protein for 4 hours at 4°C. The mTORC1 mixtures were flowed onto glutathione-conjugated agarose beads, which were then washed 10 times with HEPES buffer.

[0128] Gel electrophoresis demonstrated that distinct proteins of the expected molecular weights were isolated (FIG.13). mTOR and GST were detected by Western immunoblotting. Detected proteins show that the isolate with Rheb is highly enriched for mTORα. This data demonstrates that GST-tagged Rheb-GTP, but not control GST, can isolate mTORC1 from a previously isolated and eluted mTORC1 by the RagA- GTP / RagC-GDP method.

[0129] The method described in this Example can be used to further purify mTORC1 from any additional proteins that may have been isolated by RagA-GTP / RagC-GDP. The method can also be used to purify mTORα from a mixture of mTORα− and mTORβ- containing mTORC1 that exists in some cell-types and organ / tissue sources. The TORC1β isoform remains in the supernatant after mTORC1α is isolated by Rheb on the GST-beads. mTORC1 β isoform can be concentrated, for example, using ultrafiltration.

[0130] Antibodies used in the above Examples were purchased from Cell Signaling Technologies (CST): mTOR (#2983), Raptor (#2280), Rictor (#2114), GbetaL / LST8 (#3274), phospho-4E-BP1 Thr37 / 46 (#2855), RagA (#4357), RagC (#9480); from Santa Cruz Biotechnology: anti-GST (#sc-138); and from Sigma-Aldrich: anti-Sin1 from (#07- 2276-I).PATENT Example 9. Summary

[0131] The herein data indicates that Rag GTPases can be utilized as affinity probes to isolate endogenous mTORC1. This unique approach can be used to isolate large quantities of endogenous mTORC1 without the use of immunoaffinity reagents. It is demonstrated that the Rag GTPases, in agreement with their established role as a Raptor- binding proteins (Kim et al.2008; Sancak et al.2008), can be used as affinity probes to isolate and elute mTORC1. Furthermore, the data indicate that purified mTORC1 is isolated in its native conformation, as it is catalytically active (FIG.7A-7B).

[0132] Importantly, this method can be scaled up and applied to a variety of cellular sources, providing for isolation of mTORC1 from abundant sources like animal tissues. Although this strategy may also isolate additional Rag interacting proteins, as is likely the case even from bacterial lysates (FIG.5A, RagA band ~65 kDa), the predominant chelation-eluted proteins purified through this method correspond to known mTORC1 components (FIG.7A), indicating that this strategy on the whole is selective for mTORC1.

[0133] Moreover, the results indicate that both mTORα and mTORβ isoforms form complexes with Raptor and LST8, and that both can be readily isolated from tissue samples (FIG.11A). Secondary purification steps could be applied to achieve further purity of mTORC1, such as size-exclusion chromatography and ion-exchange methods. As shown in FIG.11A, this tool may be used not only for large prep isolation, but also for the isolation of varied isoforms of mTORC1 from animal tissues, permitting the study of variations of mTORC1 and its binding partners. REFERENCES Ash, M. R., et al. Potassium-activated GTPase reaction in the G Protein-coupled ferrous iron transporter B. J Biol Chem 285, 14594-14602, doi:10.1074 / jbc.M110.111914 (2010). Ash, M. R., et al. The cation-dependent G-proteins: in a class of their own. FEBS Lett 586, 2218-2224, doi:10.1016 / j.febslet.2012.06.030 (2012). Avruch, J., et al. Activation of mTORC1 in two steps: Rheb-GTP activation of catalytic function and increased binding of substrates to raptor. Biochem Soc Trans 37, 223-226, doi:10.1042 / BST0370223 (2009). Barbacid, M. Ras Genes. Annu Rev Biochem 56, 779-827, doi:10.1146 / annurev.bi.56.070187.004023 (1987).PATENT Bar-Peled, L., et al. A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science.340, 1100–6 (2013). Dong, J. et al. Increased adipose tissue lipolysis in dairy cows with fatty liver is associated with enhanced autophagy activity. J. Dairy Sci.105, 1731–1742 (2022). Garami, A., et al. Insulin activation of Rheb, a mediator of mTOR / S6K / 4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell.11, 1457–66 (2003). Kim, E. et al. Regulation of TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol.10, 935–45 (2008) Kjeldgaard, M., et al. The GTP binding motif: variations on a theme. FASEB J.10, 1347- 1367 (1996). Lee, M. et al. Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway. Proc Natl Acad Sci U S A 115, E5279-E5288, doi:10.1073 / pnas.1801287115 (2018). Panasyuk, G. et al. mTORbeta splicing isoform promotes cell proliferation and tumorigenesis. J. Biol. Chem.284, 30807–14 (2009). Paul, F. et al. Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners. Mol Cell Proteomics 16, 73-85, doi:10.1074 / mcp.M116.061531 (2017). Rudack, T. et al. The Role of Magnesium for Geometry and Charge in GTP Hydrolysis, Revealed by Quantum Mechanics / Molecular Mechanics Simulations. Biophys. J.103, 293– 302 (2012). Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496-1501, doi:10.1126 / science.1157535 (2008). Sancak, Y. et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290-303, doi:10.1016 / j.cell.2010.02.024 (2010). Sato, T., et al. Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein. J Biol Chem 284, 12783-12791, doi:10.1074 / jbc.M809207200 (2009). Shen, K., et al. Intersubunit Crosstalk in the Rag GTPase Heterodimer Enables mTORC1 to Respond Rapidly to Amino Acid Availability. Mol Cell 68, 821, doi:10.1016 / j.molcel.2017.10.031 (2017). Sot, B., Behrmann, et al. Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes. Proc Natl Acad Sci U S A 110, 111-116, doi:10.1073 / pnas.1201658110 (2013). Tatebe, H. et al. Evolutionary Conservation of the Components in the TOR Signaling Pathways. Biomolecules.7, 77 (2017).PATENT Vetter, I. R., et al. The guanine nucleotide-binding switch in three dimensions. Science 294, 1299-1304, doi:10.1126 / science.1062023 (2001). Wang, W., et al. Trapp Stimulates Guanine Nucleotide Exchange on Ypt1p. J. Cell Biol.151, 289–296 (2000). Wittinghofer, A. et al. Structure-function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem.80, 943–71 (2011). Zhang, B., et al. The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins. J Biol Chem 275, 25299-25307, doi:10.1074 / jbc.M001027200 (2000). *** The present invention is further described by the following claims.

Claims

PATENT CLAIMS 1. An in vitro method for isolating a binding partner of a GTPase, the method comprising: a) contacting (i) an active form of a GTPase with (ii) a sample comprising the binding partner, wherein the binding partner binds to the GTPase to form a GTPase-binding partner complex; b) capturing the GTPase-binding partner complex; and c) eluting the binding partner from the GTPase; thereby isolating the binding partner.

2. The method of claim 1, wherein the binding partner is mechanistic target of rapamycin complex 1 (mTORC1) and wherein the GTPase is a Rag heterodimer.

3. The method of claim 1, wherein the binding partner is mTORC1α and wherein the GTPase is Rheb.

4. The method of claim 1, wherein the binding partner is mechanistic target of rapamycin complex 2 (mTORC2) and wherein the GTPase is selected from Rap1 and RasC.

5. An in vitro method for isolating a mechanistic target of rapamycin complex 1 (mTORC1), the method comprising: a) contacting (i) an active Rag heterodimer comprising an affinity tag with (ii) a sample comprising mTORC1, wherein the mTORC1 binds to the Rag heterodimer to form a Rag-mTORC1 complex; b) capturing the Rag-mTORC1 complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rag heterodimer; and c) eluting the mTORC1 from the Rag heterodimer; thereby isolating mTORC1.

6. The method of claim 5, further comprising: d) contacting the mTORC1 with an active Rheb protein comprising an affinity tag, wherein the Rheb protein binds to mTORC1α to form a Rheb-mTORC1α complex;PATENT e) capturing the Rheb-mTORC1α complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rheb protein; and f) eluting the mTORC1α from the Rheb heterodimer.

7. The method of claim 5 or claim 6, further comprising analyzing the mTORC1 or the mTORC1α to identify one or more components of the complex.

8. An in vitro method for separating mTORC1α from mTORC1β, the method comprising: a) contacting (i) an active Rheb protein comprising an affinity tag with (ii) a sample comprising mTORC1α and mTORC1β, wherein the mTORC1α binds to the Rheb protein to form a Rheb-mTORC1α complex; b) capturing the Rheb-mTORC1α complex on an affinity substrate, wherein the affinity substrate comprises a binding partner of the affinity tag on the Rheb protein; c) optionally, collecting eluent comprising the mTORC1β; and d) eluting the mTORC1α from the Rheb protein; thereby separating mTORC1α from mTORC1β.

9. A method for producing a preparation of isolated GTP-bound RagA protein or RagB protein, the method comprising: a) transforming a cell with a nucleic acid comprising a sequence encoding a RagA protein or a RagB protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the cell expresses a GST-tagged RagA protein or a GST-tagged RagB protein; b) isolating the GST-tagged RagA or GST-tagged RagB protein by affinity purification with a glutathione-conjugated affinity substrate; c) releasing nucleotide from the GST-tagged RagA or GST-tagged RagB protein; and d) incubating the GST-tagged RagA or GST-tagged RagB protein with (i) GTP, GTPgS, or GDPnHP and (ii) Mg2+;PATENT thereby producing a preparation of isolated GTP-bound RagA protein or RagB protein.

10. A method for producing a preparation of isolated GDP-bound RagC protein or RagD protein, the method comprising: a) transforming a cell with a nucleic acid comprising a sequence encoding a RagC protein or a RagD protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the cell expresses a GST-tagged RagC protein or a GST-tagged RagD protein; b) isolating the GST-tagged RagC or GST-tagged RagD protein by affinity purification with a glutathione-conjugated affinity substrate; c) releasing nucleotide from the GST-tagged RagC or GST-tagged RagD protein; and d) incubating the GST-tagged RagC or GST-tagged RagD protein with GDP and Mg2+; thereby producing a preparation of isolated GDP-bound RagC protein or RagD protein.

11. The method of claim 7 or claim 10, wherein the cell is a bacterial cell.

12. A method for preparing an active Rag heterodimer, the method comprising: a) producing a preparation of isolated GTP-bound RagA protein or RagB protein, comprising: i. transforming a first cell with a nucleic acid comprising a sequence encoding a RagA protein or a RagB protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the first cell expresses a GST-tagged RagA protein or a GST-tagged RagB protein; ii. isolating the GST-tagged RagA or GST-tagged RagB protein by affinity purification with a glutathione-conjugated affinity substrate; iii. releasing nucleotide from the GST-tagged RagA or GST-tagged RagB protein; and iv. incubating the GST-tagged RagA or GST-tagged RagB protein with (i) GTP, GTPgS, or GDPnHP and (ii) Mg2+;PATENT thereby producing a preparation of isolated GTP-bound RagA protein or RagB protein; b) producing a preparation of isolated GDP-bound RagC protein or RagD protein, comprising: i. transforming a second cell with a nucleic acid comprising a sequence encoding a RagC protein or a RagD protein, operably linked to a sequence encoding glutathione S-transferase (GST), wherein the second cell expresses a GST-tagged RagC protein or a GST-tagged RagD protein; ii. isolating the GST-tagged RagC or GST-tagged RagD protein by affinity purification with a glutathione-conjugated affinity substrate; iii. releasing nucleotide from the GST-tagged RagC or GST-tagged RagD protein; and iv. incubating the GST-tagged RagC or GST-tagged RagD protein with (i) GTP, GTPgS, GDPnHP, or GDP and (ii) Mg2+; thereby producing a preparation of isolated, homogenous-nucleotide-bound RagC protein or RagD protein; and c) combining the preparation of step a) with the preparation of step b); thereby preparing an active Rag heterodimer.

13. The method of claim 12, wherein one or both of the first cell and the second cell is a bacterial cell.

14. A method for screening a candidate molecule for its ability to compete with a GTPase for binding to a binding partner of the GTPase, the method comprising: a) contacting (i) an active form of a GTPase with (ii) a sample comprising the binding partner, wherein the binding partner binds to the GTPase to form a GTPase-binding partner complex; b) capturing the GTPase-binding partner complex; c) contacting the GTPase-binding partner complex with the candidate molecule; andPATENT d) determining whether the binding partner is released from the GTPase in the presence of the candidate molecule; whereby if the binding partner is released from the GTPase, the candidate molecule is able to compete with the GTPase for binding to the binding partner.

15. The method of claim 14, wherein the GTPase is selected from a Rag heterodimer and Rheb.

16. A method for screening a candidate molecule for its ability to compete with an active Rag heterodimer for binding to mTORC1, the method comprising: a) contacting (i) the Rag heterodimer with (ii) a sample comprising mTORC1, wherein mTORC1 binds to the Rag heterodimer to form a Rag-mTORC1 complex; b) capturing the Rag-mTORC1 complex; c) contacting the Rag-mTORC1 complex with the candidate molecule; and d) determining whether mTORC1 is released from the Rag heterodimer in the presence of the candidate molecule; whereby if mTORC1 is released from the Rag heterodimer, the candidate molecule is able to compete with the Rag heterodimer for binding to mTORC1.

17. A kit comprising: (i) a composition comprising an active Rag heterodimer, wherein at least one monomer of the heterodimer comprises an affinity tag; and (ii) an affinity substrate comprising a binding partner of the affinity tag.

18. The kit of claim 17, wherein the affinity tag is GST and wherein the binding partner is glutathione.

19. The kit of claim 17, wherein the affinity substrate is agarose beads.

20. The kit of claim 17, wherein the Rag heterodimer is bound to the affinity substrate via the affinity tag.

21. The kit of claim 17, further comprising an elution buffer, wherein the elution buffer comprises a magnesium chelating agent and / or a monovalent chloride salt.

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