Detection of conformationally altered proteins and prions
The invention provides methods and kits for detecting conformationally altered proteins and prions in a sample. In one embodiment, the conformationally altered proteins and prions are associated with amyloidogenic diseases.
1 . A method for detecting ββ-sheet conformation of insoluble proteins or prions in a sample comprising:
(a) reacting the sample with one or more α-helix or random coil conformational probes that interact with ββ-sheet conformation insoluble proteins or prions in the sample and thereby (i) undergo a conformational conversion to a predominately to ββ-sheet conformation, and (ii) form detectable aggregates with the β-sheet conformation insoluble proteins or prions in the sample; and
(b) detecting levels of detectable aggregates, wherein levels of detectable aggregates correlate to the levels of ββ-sheet conformation insoluble proteins or prions in the sample.
2 . A method of claim 1 , wherein probe termini are bound to moieties that are optically detectable when the probes form detectable aggregates with the ββ-sheet conformation insoluble proteins or prions in the sample.
3 . A method of claim 2 , wherein the moieties are fluorophores.
4 . A method of claim 1 , wherein probe termini are bound to radionucleotide moieties that are detectable when the probes form detectable aggregates with the ββ-sheet conformation insoluble proteins or prions in the sample.
5 . A method of claim 1 , wherein the probes comprise at least two amino acid sequences that are complimentary to amino acid sequences of the ββ-sheet conformation insoluble proteins or prions.
6 . A method of claim 1 , wherein one or more of the probes comprise at least two amino acid sequences that are homologous to amino acid sequences of the ββ-sheet conformation insoluble proteins or prions.
7 . A method claim 6 , wherein one or more of the probes is a palindromic probe.
8 . A method of claim 1 , wherein the ββ-sheet conformation insoluble proteins or prions are selected from the group consisting of low-density lipoprotein receptor, cystic fibrosis transmembrane regulator, Huntingtin, Abeta peptide, prions, insulin-related amyloid, hemoglobin, alpha synuclein, rhodopsin, crystallins, and p53.
9 . A method of claim 1 , where one or more probes is a palindromic 33_mer comprising amino acid sequences that are homologous to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO: 1 or 29).33_mer palindrome
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
10 . A method of claim 1 , wherein one or more probes is a palindromic 33_mer comprising amino acid sequences that are equivalent to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO: 1 or 29).33 _mer palindrome
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
11 . A method of claim 1 , wherein one or more probes is a palindromic 33_mer comprising amino acid sequences that are between about 70% to about 90% identical to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO:1 or 29).33_mer palindrome
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
12 . A method of claim 1 , wherein one or more probes is a probe comprising amino acid sequences that are homologous to amino acids 1-40 of the Abeta peptide Nref 00111747
(human)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG
SEQ ID NO: 4
GVV.
13 . A method of claim 1 , wherein one or more probes comprise amino acid sequences that are equivalent to amino acids 1-40 of the Abeta peptide (SEQ ID NO:4).
(SEQ ID NO:4).
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV
GGVV.
14 . A method of claim 1 , wherein one or more probes comprise amino acid sequences that are between about 70% to about 90% identical to amino acids 1-40 of the A.beta peptide (SEQ ID NO:4)
(SEQ ID NO:4)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG
GVV.
15 . A method of claim 1 , wherein one or more probes comprise an amino acid sequence that has a helix-loop-helix conformation found in polylysine and that is equivalent to
SEQ ID NO: 8.
KKKKKKKKKKKKKKKKKKKKKKKKKKK.
16 . A method of claim 1 , wherein one or more probes comprise an amino acid sequence that has a helix-loop-helix conformation found in polylysine and that is homologous to
SEQ ID NO: 8.
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK.
17 . A method of claim 1 , wherein one or more probes comprise an amino acid sequence that has a helix-loop-helix conformation found in polylysine and that is equivalent to
SEQ ID NO: 8
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK.
18 . A method of claim 1 , wherein one or more probes comprise an amino acid sequence that has a helix-loop-helix conformation found in polylysine and that is between about 70% to about 90% identical to SEQ ID NO: 8.
SEQ ID NO: 8.
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK.
19 . A method of claim 1 , wherein one or more probes comprise amino acid sequences that are homologous to amino acids 104-122 of wild-type (wt) TSE (SEQ ID NO:10)
(SEQ ID NO:10)
KPKTNLKHVAG AAAAGAVV .
20 . A method of claim 1 , wherein one or more probes comprise amino acid sequences that are equivalent to amino acids 104-122 of wild-type (wt) TSE (SEQ ID NO:10).
(SEQ ID NO:10).
KPKTNLKHVAG AAAAGAVV
21 . A method of claim 1 , wherein one or more probes comprise amino acid sequences that are between about 70% to about 90% identical to amino acids 104-122 of wild-type
(SEQ ID NO:10)
KPKTNLKHVAGAAAAGAVV.
22 . A method of claim 1 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; and (b) is destabilized and noninfectious;
and (c) has an amino acid sequence that is homologous to SEQ ID NO: 10
SEQ ID NO: 10
KPKTNLKHVAGAAAAGAVV.
23 . A method of claim 1 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; (b) is destabilized and noninfectious; and
(c) has an amino acid sequence that is equivalent to SEQ ID NO: 10
SEQ ID NO: 10
KPKTNLKHVAGAAAAGAVV.
24 . A method of claim 1 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; (b) is destabilized and noninfectious; and
(c) has an amino acid sequence that is between about 70% to about 90% identical to SEQ ID NO: 10
SEQ ID NO: 10
KPKTNLKHVAGAAAAGAVV.
25 . The method of claim 1 , wherein the probes comprise an extrinsic fluor.
26 . The method of claim 25 , wherein the extrinsic fluor is pyrene.
27 . A method of claim 1 , further comprising reacting the sample and probes prior to detecting with a probe that limits the formation of detectable aggregates to detectable but non-infectious levels.
28 . A method of claim 1 , wherein levels of detectable aggregates are compared to levels of ββ-sheet conformation insoluble proteins or prions associated with amyloidogenic diseases.
29 . A method of claim 1 , wherein the ββ-sheet conformation insoluble proteins or prions form amyloid plaques or amyloid deposits associated with amyloidogenic diseases.
30 . A method of claim 1 , wherein the sample is disaggregated prior to reaction with the probe.
31 . A method of claim 1 , wherein the sample is a tissue sample or is a liquid biological material obtained from spinal fluid, saliva, urine or other bodily fluids.
32 . A method of claim 1 , wherein excimers are formed by reacting one or more α-helix or random coil conformational probes with ββ-sheet conformation insoluble proteins or prions in the sample.
33 . A kit comprising one or more α-helix or random coil conformational probes that interact with β-sheet conformation insoluble proteins or prions in a sample and thereby (a) undergo a conformational conversion to a predominately to ββ-sheet conformation, and (b) form detectable aggregates with the ββ-sheet conformation insoluble proteins or prions in the sample, wherein levels of detectable aggregates correlate to the levels of ββ-sheet conformation insoluble proteins or prions in the sample.
34 . A kit of claim 33 , wherein probe termini are bound to moieties that are optically detectable when the probes form detectable aggregates with ββ-sheet conformation insoluble proteins or prions in a sample.
35 . A kit of claim 34 , wherein the moieties are fluorophores.
36 . A kit of claim 33 , wherein probe termini are bound to radionuclide moieties that are detectable when the probes form detectable aggregates with ββ-sheet conformation insoluble proteins or prions in a sample.
37 . A kit of claim 33 , wherein the probes comprise at least two amino acid sequences that are complementary to amino acid sequences of ββ-sheet conformation insoluble proteins or prions.
38 . A kit of claim 33 , wherein one or more of the probes comprise at least two amino acid sequences that are homologous to amino acid sequences of ββ-sheet conformation insoluble proteins or prions.
39 . A kit of claim 33 , wherein one or more of the probes comprise an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, 22, 23, 24, 25 or 27.
40 . A kit of claim 33 , wherein the ββ-sheet conformation insoluble proteins or prions are selected from the group consisting of low-density lipoprotein receptor, cystic fibrosis transmembrane regulator, Huntingtin, Abeta peptide, prions, insulin-related amyloid, hemoglobin, alpha synuclein, rhodopsin, crystallins, and p53.
41 . A kit of claim 33 , where one or more probes is a palindromic 33_mer comprising amino acid sequences that are homologous to amino acids 122-104 and 109-122 of the human or murine PrP SC protein (SEQ ID NO: 1 or 29)
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
42 . A kit of claim 33 , wherein one or more probes is a palindromic 33_mer comprising amino acid sequences that are equivalent to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO: 1 or 29).
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
43 . A kit of claim 33 , wherein one or more probes is a palindromic 33_mer comprising amino acid sequences that are between about 70% to about 90% identical to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO: 1 or 29).
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
44 . A kit of claim 33 , wherein one or more probes is a probe comprising amino acid sequences that are homologous to amino acids 1-40 of the Abeta peptide (SEQ ID NO:
4). DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
45 . A method of claim 1 , wherein one or more probes comprise amino acid sequences that are equivalent to amino acids 1-40 of the Abeta peptide (SEQ ID NO:4).
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.
46 . A kit of claim 33 , wherein one or more probes comprise amino acid sequences that are between about 70% to about 90% identical to amino acids 1-40 of the Abeta peptide (SEQ ID NO:4).
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.
47 . A kit of claim 33 , wherein one or more probes comprise an amino acid sequence that is equivalent or homologous to SEQ ID NO: 9 or 20.
48 . A kit of claim 33 , wherein one or more probes comprise an amino acid sequence that has a helix-loop-helix conformation found in polylysine and that is homologous to SEQ ID NO: 8.
49 . A kit of claim 33 , wherein one or more comprise an amino acid sequence that has a helix-loop-helix conformation found in polylysine and that is equivalent to SEQ ID NO: 8.
50 . A kit of claim 33 , wherein one or more probes comprise an amino acid sequence that has a helix-loop-helix conformation found in polylysine and that is between about 70% to about 90% identical to SEQ ID NO: 9.
51 . A kit of claim 33 , wherein one or more probes comprise amino acid sequences that are homologous to amino acid sequences 104-122 of wild-type (wt) TSE (SEQ ID NO: 10).
KPKTNVKHVAG AAAAGAVV .
52 . A kit of claim 33 , wherein one or more probes comprise amino acid sequences that are equivalent to amino acid sequences 104-122 of wild-type (wt) TSE (SEQ ID NO: 10).
KPKTNVKHVAG AAAAGAVV .
53 . A kit of claim 33 , wherein one or more probes comprise amino acid sequences that are between about 70% to about 90% identical to amino acid sequences 104-122 of wild-type (wt) TSE (SEQ ID NO: 10).
KPKTNVKHVAG AAAAGAVV .
54 . A kit of claim 33 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; (b) is destabilized and noninfectious; and
(c) has an amino acid sequence that is homologous to SEQ ID NO: 10.
55 . A kit of claim 33 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; (b) is destabilized and noninfectious; and
(c) has an amino acid sequence that is equivalent to SEQ ID NO: 10.
56 . A kit of claim 33 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; (b) is destabilized and noninfectious; and
(c) has an amino acid sequence that is between about 70% to about 90% identical to SEQ ID NO: 10.
57 . A kit of claim 33 , wherein the probes comprise an extrinsic fluor.
58 . A kit of claim 57 , wherein the extrinsic flour is pyrene.
59 . A kit of claim 33 , further comprising a pendant probe that limits the formation of detectable aggregates to detectable but non-infectious levels.
60 . A method of diagnosing whether a subject suffers from, or is predisposed to, a disease associated with conformationally altered proteins or prion comprising:
(a) obtaining a sample from the subject;
(b) reacting the sample with one or more α-helix or random coil conformational probes that interact with ββ-sheet conformation insoluble proteins or prions in the sample and thereby (i) undergo a conformational conversion to a predominately to ββ-sheet conformation, and (ii) form detectable aggregates with the ββ-sheet conformation insoluble proteins or prions in the sample; and
(c) detecting levels of detectable aggregates, wherein levels of detectable aggregates correlate to the amount of ββ-sheet conformation insoluble proteins or prions in, and level of infectiousness of, the sample and indicate whether the subject suffers from, or is predisposed to, a disease associated with ββ-sheet conformation insoluble proteins or prions.
61 . A method of claim 60 , wherein probe termini are bound to moieties that are optically detectable when the probes form detectable aggregates with the ββ-sheet conformation insoluble proteins or prions in the sample.
62 . A method of claim 61 , wherein the moieties are fluorophores.
63 . A method of claim 60 , wherein probe termini are bound to radionuclide moieties that are detectable when the probes form detectable aggregates with the ββ-sheet conformation insoluble proteins or prions in the sample.
64 . A method of claim 60 , wherein the probes comprise at least two amino acid sequences that are complimentary to amino acid sequences of the ββ-sheet conformation insoluble proteins or prions.
65 . A method of claim 60 , wherein one or more of the probes comprise at least two amino acid sequences that are homologous to amino acid sequences of the ββ-sheet conformation insoluble proteins or prions.
66 . A method claim 60 , wherein one or more of the probes comprise an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, 22, 23, 24, 25 or 27.
67 . A method of claim 60 , wherein the ββ-sheet conformation insoluble proteins or prions are selected from the group consisting of low-density lipoprotein receptor, cystic fibrosis transmembrane regulator, Huntingtin, Abeta peptide, prions, insulin-related amyloid, hemoglobin, alpha synuclein, rhodopsin, crystallins, transthyretin, gelsolin, cystatins and p53.
68 . A method of claim 60 , where one or more probes is a palindromic 33_mer comprising amino acid sequences that are homologous to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO: 1 or 29).
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
69 . A method of claim 60 , wherein one or more probes is a palindromic 33_mer comprising amino acid sequences that are equivalent to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO:1 or 29)
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
70 . A method of claim 60 , wherein one or more probes is a palindromic 33_mer comprising amino acid sequences that are between about 70% to about 90% identical to amino acids 122-104 and 109-122 of the PrP SC protein (SEQ ID NO: 1 or 29).
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAA GAMHKMNTKPKMKHMAG AAAAGAVV (human)
71 . A method of claim 60 , wherein one or more probes is a probe comprising amino acid sequences that are homologous to amino acids 1-40 of the Abeta peptide (SEQ ID NO:4)
(SEQ ID NO:4)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG
GVV.
72 . A method of claim 60 , wherein one or more probes comprise amino acid sequences that are equivalent to amino acids 1-40 of the Abeta peptide (SEQ ID NO:4)
(SEQ ID NO:4)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG
GVV
73 . A method of claim 60 , wherein one or more probes comprise amino acid sequences that are between about 70% to about 90% identical to amino acids 1-40 of the Abeta peptide
(SEQ ID NO:4).
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV
GGVV
74 . A method of claim 60 , wherein one or more probes comprise an amino acid sequence that is an oligo or polylysine.
75 . A method of claim 74 , wherein said probe is homologous to SEQ ID NO: 8.
76 . A method of claim 60 , wherein said probe is equivalent to SEQ ID NO: 8.
77 . A method of claim 60 , wherein one or more probes comprise an amino acid sequence that has a helix-loop-helix conformation found in lysine and that is between about 70% to about 90% identical to oligo- or polylysine.
78 . A method of claim 61 , wherein one or more probes comprise amino acid sequences that are homologous or equivalent to amino acids 104-122 of wild-type (wt) TSE (SEQ ID NO:10).
79 . A method of claim 60 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; (b) is destabilized and noninfectious; and
(c) has an amino acid sequence that is homologous or equivalent to SEQ ID NO: 10.
80 . A method of claim 61 , wherein one or more probes comprise an amino acid sequence that: (a) is a selectively mutated TSE sequence; (b) is destabilized and noninfectious; and
(c) has an amino acid sequence that is between about 70% to about 90% identical to SEQ ID NO: 10.
81 . A method of claim 60 , wherein the probes comprise an extrinsic fluor.
82 . The method of claim 60 , wherein the extrinsic flour is pyrene.
83 . A method of claim 60 , further comprising reacting the sample and probes prior to detecting with a pendant probe that limits the formation of detectable aggregates to detectable but non-infectious levels.
84 . A method of claim 60 , wherein levels of detectable aggregates are compared to levels of ββ-sheet conformation insoluble proteins or prions associated with amyloidogenic diseases.
85 . A method of claim 60 , wherein the ββ-sheet conformation insoluble proteins or prions form amyloid plaques or amyloid deposits associated with amyloidogenic diseases.
86 . A method of claim 60 , wherein the sample is disaggregated prior to reaction with the probe.
87 . A method of claim 60 , wherein the sample is a tissue sample or is a liquid biological material obtained from spinal fluid, saliva, urine or other bodily fluids.
88 . A method of claim 60 , wherein exi_mers are formed by reacting one or more α-helix or random coil conformational probes with ββ-sheet conformation insoluble proteins or prions in the sample.
89 . A palindromic peptide probe comprising three peptide sections, a first peptide section, a second peptide section and a third peptide section, said first and said third sections comprising peptide sequences each of which comprises at least 5 amino acids identical to a peptide fragment from a target insoluble protein which is responsible for ββ-sheet formation in said target insoluble protein and wherein at least a portion of said first peptide section is a palindrome of at least a portion of said third peptide section, said first peptide section or said third peptide section being identical to at least a five amino acid peptide sequence in said peptide fragment from said target insoluble protein, said second peptide sequence comprising between 1 and 10 amino acid units one of which is a proline residue.
90 . The probe according to claim 89 wherein said first and said third sections are endcapped with hydrophobic amino acids which can be chemically modified or complexed to accommodate a chemical moiety capable of being measured.
91 . The probe according to claim 90 wherein said chemical moiety is a chromophore and both said first and third peptide sections of said probe comprise said chromophore.
92 . The probe according to claim 90 wherein said chromophore is selected from the group consisting of pyrene, tryoptophan, fluresceing rhodamine.
93 . The probe according to claim 92 which is in the form of an excimer.
94 . The probe according to claim 89 wherein said second proline section comprises between 1 and 5 amino acid residues all of which are proline residues.
95 . The probe according to claim 89 wherein said target peptide is selected from the group consisting of low-density lipoprotein receptor, cystic fibrosis transmembrane regulator, Huntingtin, Abeta peptide, prions, insulin-related amyloid, hemoglobin, alpha synuclein, rhodopsin, crystallins, transthyretin, gelsolin, cystatins and p53.
96 . The probe according to claim 89 wherein said first peptide section and said third peptide section consist of identical amino acids.
97 . The probe according to claim 89 wherein said first and said second peptide sections each comprise about 10 to about 25 amino acid residues.
98 . The palindromic probe according to claim 89 selected from the group consisting of SEQ ID NO: 1, 18, 23, 25, 27 and 29.
99 . The method according to claim 60 wherein said disease is Alzheimer's Disease, Prion diseases, Creutzfeld Jakob disease, scrapie and bovine spongiform encephalopathy (PrP Sc ); ALS (SOD and neurofilament); Pick's disease; Parkinson's disease, Frontotemporal dementia; Diabetes Type II (Amylin); Multiple myeloma—plasma cell dyscrasias; Familial amyloidotic polyneuropathy; Medullary carcinoma of thyroid;
Chronic renal failure, Congestive heart failure, Senile cardiac and systemic amyloidosis (Transthyretin), Chronic inflammation, Atherosclerosis, Familial amyloidosis, or Huntington's disease.