SYNTHETIC PROTEIN CIRCUITS DETECTING SIGNAL TRANSDUCER ACTIVITY
Disclosed herein include methods, compositions, and kits suitable for use in detecting the activation level of a signal transducer. In some embodiments, there are provided synthetic protein circuits wherein recruitment of synthetic protein circuit components to an association location upon activation of a signal transducer generates an active effector protein. The effector protein can be configured to carry out a variety of functions when in an active state, such as, for example, inducing cell death. Methods of treating a disease or disorder characterized by aberrant signaling are provided in some embodiments.
1 .- 49 . (canceled)
50 . A method of treating a disease or disorder characterized by an aberrant signaling of one or more signal transducers comprising:
expressing a synthetic protein circuit in a cell of a subject in need thereof, the synthetic protein circuit comprising:
a first polypeptide comprising a first signal transducer binding domain and a first part of a first protease domain, wherein the first signal transducer binding domain is capable of binding a first signal transducer of the cell to form a first signal transducer-bound polypeptide;
a second polypeptide comprising a second signal transducer binding domain and a second part of the first protease domain, wherein the second signal transducer binding domain is capable of binding a second signal transducer of the cell to form a second signal transducer-bound polypeptide, wherein the first part of the first protease domain and the second part of the first protease domain have weak association affinity, and wherein the first part of the first protease domain and the second part of the first protease domain are capable of associating with each other to constitute a first protease capable of being in a first protease active state when the first signal transducer and the second signal transducer are in close proximity at an association location; and
an effector protein comprising a first cut site the first protease in the first protease active state is capable of cutting to change the effector protein to an effector active state, or an effector inactive state, which correlates with an aberrant signaling of the first signal transducer and/or the second signal transducer, and wherein the effector protein in the effector active state, or the effector inactive state, is capable of changing a state of the cell, thereby treating a disease or disorder characterized by the aberrant signaling of the first signal transducer and/or the second signal transducer.
51 . The method of claim 50 , wherein the first signal transducer binding domain of the first polypeptide and the second signal transducer binding domain of the second polypeptide are identical, and/or wherein the first signal transducer and the second signal transducer are identical.
52 . The method of claim 50 , wherein the first signal transducer when in a first signal transducer active state is capable of being localized at the association location.
53 . The method of claim 50 , wherein the second signal transducer when in a second signal transducer active state is capable of being localized at the association location.
54 . The method of claim 50 , wherein both the first signal transducer when in a first signal transducer active state and the second signal transducer when in a second signal transducer active state are capable of being localized at the association location.
55 . The method of claim 50 , wherein the first signal transducer when in a first inactive state, the second signal transducer when in a second inactive state, or both, are capable of being localized at the association location.
56 . The method of claim 50 , wherein the first signal transducer and/or the second signal transducer regulate cell survival, cell growth, cell proliferation, cell adhesion, cell migration, cell metabolism, cell morphology, cell differentiation, apoptosis, or any combination thereof.
57 . The method of claim 50 , wherein the first signal transducer, the second signal transducer, or both comprise AKT, PI3K, MAPK, p44/42 MAP kinase, TYK2, p38 MAP kinase, PKC, PKA, SAPK, ELK, JNK, cJun, RAS, Raf, MEK 1/2, MEK 3/6, MEK 4/7, ZAP-70, LAT, SRC, LCK, ERK 1/2, Rsk 1, PYK2, SYK, PDK1, GSK3, FKHR, AFX, PLCγ, PLCy, NF-kB, FAK, CREB, αIIIβ3, FcεRI, BAD, p70S6K, STAT1, STAT2, STAT3, STAT5, STAT6, or any combination thereof.
58 . The method of claim 50 , wherein the first signal transducer, the second signal transducer, or both comprise a RAS protein, and wherein the RAS protein is KRAS, NRHAS, HRAS, or any combination thereof.
59 . The method of claim 50 , wherein the first signal transducer binding domain and/or the second signal transducer binding domain comprise a RAS binding domain (RBD) and/or RAS association domain (RAD).
60 . The method of claim 50 , wherein the disease or disorder is characterized by an aberrant signaling of the first signal transducer.
61 . The method of claim 50 , wherein the disease or disorder is characterized by an aberrant signaling of the first signal transducer and an aberrant signaling of the second signal transducer.
62 . The method of claim 50 ,
wherein the disease or disorder is characterized by an aberrant signaling of a RAS protein,
wherein the disease or disorder is a cancer,
wherein the disease or disorder is a RASopathy selected from the group comprising Neurofibromatosis Type 1, Noonan syndrome, Noonan syndrome with multiple lentigines (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, or any combination thereof,
wherein the disease is a neurological disease or a neurodegenerative disease,
wherein the disease is an autoimmune disease, or
wherein the disease is infectious disease.
63 . The method of claim 50 , further comprising administering a prodrug.
64 . The method of claim 63 , wherein the prodrug is 5-fluorocytosine (5-FC) or ganciclovir.
65 . The method of claim 50 , wherein the expressing comprises administering a nucleic acid encoding the synthetic protein circuit.
66 . The method of claim 50 , wherein the expressing comprises administering two or more nucleic acids, and wherein the two or more nucleic acids encode the synthetic protein circuit.
67 . The method of claim 65 , wherein the nucleic acid comprises at least one regulatory element for expression of the synthetic protein circuit.
68 . The method of claim 65 , wherein the nucleic acid comprises a vector,
wherein the vector comprises a adenovirus vector, an adeno-associated virus vector, an Epstein-Barr virus vector, a Herpes virus vector, an attenuated HIV vector, a retroviral vector, a vaccinia virus vector, or any combination thereof,
wherein the vector comprises an RNA viral vector,
wherein the vector is derived from one or more negative-strand RNA viruses of the order Mononegavirales, and/or
the vector is a rabies viral vector.
69 . The method of claim 65 , wherein the administering comprises aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracisternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, and/or intradermal injection, or any combination thereof.