JPWO2006064819A1 - Method for diagnosing diseases caused by protein morphological changes using neutron scattering - Google Patents

Abstract

The present invention results from a change in the shape of a test protein, characterized by irradiating a test protein dissolved or dispersed in heavy water with cold neutrons and measuring the form of the test protein from the scattering of the cold neutrons The present invention relates to a method for diagnosing a disease. According to the present invention, it was possible to observe the morphological change of a protein in an aqueous solution while being non-invasive and natural with respect to the protein, and to elucidate the relationship between the morphological change and a disease. As a result, it became possible to diagnose diseases caused by protein morphological changes and to screen for drugs.

Description

  The present invention relates to a method for diagnosing a disease caused by a change in the form of a protein and a method for screening a drug by observing the change in the form of the protein present in the living body in a non-invasive and natural manner.

  In recent years, proteosome is involved in many fibrotic diseases such as Parkinson's disease, Alzheimer's disease, etc., and protein fibrosis due to abnormalities or structural abnormalities of microtubule-binding proteins tau and synuclein, and degradation of unwanted proteins in cells It has been found that protein aggregate formation, neurofibrillary cell degeneration, and cell death caused by gene abnormality of UCH-L1 (ubiquitin carboxyl-terminal hydrolase), which controls the intracellular recycling system of ubiquitin involved in the system, are deeply involved. It was. Therefore, it is considered that morphological changes such as protein fibrillation and aggregate formation in the living body are deeply involved in the cause, progress, healing process, and the like of various diseases.

With regard to conventional protein structure analysis, various techniques for analyzing the structure of crystallized proteins have been developed. By this technique, substantial morphological changes of proteins in living bodies (eg, oligomer formation by self-assembly) Etc.) is very difficult to predict. Thus, several techniques for analyzing nano-level protein structures in aqueous solutions have been developed. Of these, the X-ray small-angle scattering method can observe the aqueous solution structure of proteins. However, protein damage due to X-ray irradiation in water is inevitable, and intermolecular due to radical generation and polymerization reactions. Covalent bond formation occurs. Therefore, this method is not suitable for analysis of complex structure by intermolecular interaction. According to the nuclear magnetic resonance method (NMR), analysis is possible if it is a single protein molecule having a molecular weight of up to about 30,000, but if a protein or complex having a molecular weight higher than that is formed, ¹³ C or Analysis is difficult without special molecular modification using isotopes such as ¹⁵ N. Atomic force microscopy (AFM) is a technique in which protein is adsorbed on the mica surface in water, the surface of the protein is scanned with a microprobe, and its shape is observed. There is a problem that modification is required.

  Therefore, the present invention observes the morphological change of the protein in the aqueous solution while keeping it non-invasive and natural with respect to the protein, elucidates the relationship between the morphological change and the disease, An object is to provide a screening method.

  Therefore, the present inventor has observed the protein in the aqueous solution by using the small-angle neutron scattering method. When the cold neutron is irradiated to the protein in the heavy water, the protein does not have any chemical modification or mechanical action in the aqueous solution. The protein structure can be observed in a non-invasive and natural manner, and there is a correlation between the form of a normal protein and an abnormal protein in an aqueous solution and the disease. The present invention has been completed by finding out what can be done.

That is, the present invention relates to a change in the form of a test protein, characterized by irradiating a test protein dissolved or dispersed in heavy water with cold neutrons and measuring the form of the test protein from the scattering of the cold neutrons. The present invention provides a method for diagnosing a disease caused by the disease.
The present invention also relates to a pharmaceutical screening method using a target protein treated with a test substance or a target protein isolated from an animal to which the test substance is administered, the target protein dissolved or dispersed in heavy water. The present invention provides a screening method for a therapeutic drug for a disease caused by morphological change of a target protein, characterized by irradiating cold neutrons and measuring the form of the target protein from the scattering of the cold neutrons.

  According to the method of the present invention, the structure of the protein in the aqueous solution, which is similar to the state existing in the living body, can be observed without being invasive and natural. The relationship between the change and the disease can be observed, the initial state, progress state, healing process, etc. of various diseases can be accurately diagnosed, and medicines can be screened.

It is a conceptual diagram of the observation method of the protein structure in the heavy water solvent by neutron small angle scattering. This is a theoretical formula for obtaining a scattering vector q and scattering intensity I (q) of a core-shell type protein structure. It is a figure which shows the crystal structure of normal type UCH-L1. (Analyzed by SWISS-MODEL from crystal structure analysis data of UCH-L3, which has hydrolase activity (50% amino acid sequence matches) as in UCH-L1, and is widely distributed in the living body) It is a figure which shows the measurement result of the normal curve from the monomer and dimer of globular protein UCH-L1, and an actual normal type UCH-L1. I _e (q); measurement (●), I _c (q); theoretical curve, (−−−), monomer; (−−−), dimer. It is a theoretical curve at the time of setting it as a UCH-L1 spheroid. a. A theoretical curve of a rotating disk body (minor axis; a, major axis; b, (= c)) in which the minor axis a of the spherical monomer is gradually changed, b. A theoretical curve of a rotating disc body (minor axis; a, major axis; b, (= c)) in which the minor axis a of the spherical dimer is gradually changed, c. It is a theoretical curve of a spindle (minor axis; a, (= b), major axis; c) in which the minor axis a, (= b) of a spherical dimer is gradually changed. It is a figure which shows fitting by the neutron scattering curve and theoretical curve of normal type UCH-L1 and an amino acid substitution product. a. Normal type UCH-L1, I _e (q); Measurement (●), I _c (q); Theoretical curve, (−−−), monomer (a, 29Å, b, (= c), 52Å); (− -), Dimer, b. I93M substitution, I _e (q); measurement (●), I _c (q); theoretical curve, (---), monomer (a, 20Å, b, (= c), 62Å); (---) Dimer, c. S18Y substitution, I _e (q); measurement (●), I _c (q); theoretical curve, (---), monomer (a, 43Å, b, (= c), 43Å); (---) Dimer, d. I93M + S18Y substitution, I _e (q); measurement (●), I _c (q); theoretical curve, (−−−), monomer (a, 31Å, b, (= c), 50Å); (−−−) , Dimer. It is a figure which shows the secondary structure change by the circular dichroism measurement of normal type UCH-L1 and an amino acid substitution product. It is a figure which shows the form change and functional change by normal type UCH-L1 and amino acid substitution. It is a figure which shows the single molecule | numerator (spherical cluster formation by self-assembly) structure of microtubule binding protein tau, and the fibrosis by SS bond formation. Fibrosis tau (◯); Tau self-assembly by reduction and cleavage of SS bond (●). As a comparison, the theoretical scattering curve from a globular protein having a molecular weight equivalent to that of tau is shown as a black solid line.

  In the diagnostic method of the present invention, the test protein is dissolved or dispersed in heavy water. In the living body, proteins are dissolved in cells or body fluids. Therefore, in the present invention, it is possible to observe the natural state of the protein in the living body. Here, examples of the test protein include all proteins considered to be involved in various diseases, such as enzymes, membrane proteins, receptor proteins, structural proteins, and the like. More specific examples include UCH-L1, microtubule binding protein tau and the like.

  These proteins may be proteins isolated from living organisms or may be proteins synthesized using genes derived from living organisms. The synthesis of a protein using a gene can be performed by a known method as long as the structure of the gene is known.

  The protein concentration in heavy water is not particularly limited, but 10 μM or more is sufficient. Preferably it is 10 μM to 100 mM, more preferably 10 μM to 10 mM.

  Cold neutrons can be converted from neutrons by known means. That is, a particle beam such as a proton accelerated by an accelerator is applied to a target made of a heavy element (for example, uranium, tungsten, mercury etc), causing nuclear fragmentation, obtaining pulsed neutrons, which are used as moderators ( Liquid hydrogen, liquid deuterium, solid methane, etc.). Similarly, neutrons generated from nuclear reactors as uranium decays can be converted to cold neutrons in the same way (New Polymer Experiment 6 Polymer Structure (2) Scattering Experiments and Morphology, p282 Kyoritsu Publishing Co., Ltd.) reference). Here, the wavelength of the cold neutron is about 0.5 to 1.5 nm.

Even when the cold neutrons are irradiated to protein nuclei, they are only scattered and do not crush the nuclei, so there is almost no damage to the proteins. The scattering ability of cold neutrons to atoms is determined by the scattering length density of the atoms. In particular, deuterium ( ² H, deuterium; D) has a large positive scattering length, whereas that of light hydrogen ( ¹ H) is negative. When this is utilized, cold neutrons are relatively easily scattered by hydrogen atoms ( ¹ H) in proteins and easily absorbed by heavy water (D ₂ O) as a solvent. Therefore, when protein is dissolved and dispersed in heavy water, a large contrast is produced and the protein structure can be observed (neutron small angle scattering method). The analysis range ranges from several to submicrons. This conceptual diagram is shown in FIG.

  Here, the neutron small angle scattering method will be described. The generated cold neutron is irradiated onto the sample through a slit-type neutron conduit. A two-dimensional detector is used to detect scattered light generated from the sample. The camera length can be arbitrarily changed by moving through the vacuum chamber according to the q (momentum change) range to be measured. Although it is necessary to adjust the collimation of the slit portion according to the camera length, this mechanism can obtain accurate data in a wide q range. It is also possible to perform measurements at different camera lengths, i.e. q ranges, and superimpose them into a single composite curve. The cell portion is preferably devised so that a temperature control, a sample changer, or the like can be incorporated. Special cell units such as high temperature, low temperature, and high pressure can also be used.

As the sample cell, it is common to use a square or drum-shaped quartz container. The neutron beam passes through the boron-free quartz glass with almost no absorption, so that an easy-to-handle cell having a cell container wall thickness of about 1 mm can be used. It is common that the thickness is 1 to 10 mm with 100% D ₂ O. The thinner the cell thickness, the weaker the scattering and the longer integration is required. It is desirable to clean the cell sufficiently by ultrasonic cleaning or the like.

  The measurement of scattered light is preferably performed by measuring transmittance separately from the scattering measurement, and subtracting the background scattering due to the cell, the solvent, and the like using the measured value. If the composition is known, it can be subtracted by calculation, but it is preferable to measure sufficiently to the wide-angle side and experimentally obtain and subtract this background. Since point beams are used in the small-angle neutron scattering method, there is usually no need for desmearing. In the case of an isotropic sample, a circular average of two-dimensional data obtained by a two-dimensional detector is taken and converted into one-dimensional data by a computer.

  Since the form of the protein can be observed by computer processing of scattered light, a disease can be diagnosed by observing the change in the form, for example, the change in shape and / or size. The analysis method used for diagnosis is to predict the shape of various proteins as shown in Tables 1, 2, and 3, calculate the scattering vector, q = (4π / λ) sin θ and the scattering intensity, and which shape is the most It can be analyzed whether it corresponds to the experimental value.

(1) Hideki Matsuoka, “Application to colloids, micelles and polymer solutions”, Journal of the Crystallographic Society of Japan, 41, 269 (1999).
(2) J. Org. S. Higgins, H .; G. Benoiit, “Polymerand Neutron Scattering”, Oxford, (1994).
(3) O.I. Kratky, O .; Glalter, ed. “Small-angle X-ray Scattering”, Chap. 12, Academic 1982.
(4) T.W. Neugebauer, Ann. Phy. , 42, 509 (1943).
(5) P.I. Debye, J .; Phys. Colloid Chem. 51, 18 (1948).
(6) H. Benoit, J.A. Polym. Sci. 11, 507 (1953).
(7) K. Kajiwara, W. et al. Burchard, M .; Gordon, Br. Polym. J. et al. 2,110 (1970).
Other references F. Casassa, G .; C. Berry, J .; Polym. Sci. , A-2, 4, 881 (1966).
E. F. Casassa, J. et al. Polym. Sci. A. 3,605 (1965).
O. B. Ptyysyn, Zhurnal Fizicheskoi Kimii, 31.1091 (1957).
A. Peterlin, J. et al. Chem. Phys. , 23, 2464 (1955).

  In the region where the scattering vector q is large (q to 1 / Rg, Rg is the radius of rotation of the center of gravity), Guinier's law (reference: A. Gunier, G. Fournet, Small Angle Scattering of X-rays, Wiley, New). According to York, 1955.), the relationship between the scattering vector q and the scattering intensity I (q) can be expressed as I (q) = I (0) exp (−1/3 Rg2q2), as shown in Table 4. The protein shape can also be estimated by such calculation. I (0) is a value obtained by extrapolating q to 0. When the scattering intensity is converted into an absolute value, this value depends on the molecular weight (reference: Jacrot and Zaccai (Biopolymers, 20 (1981), 2413-2426)).

  In addition, an analysis method as shown in FIG. 2 (J. Marignan, P. Basserau, and P. Delord, J. Phys. Chem., 90, etc.) is also used for complex protein structures that form a core at the center of the protein. 645 (1986)), shape parameters can be determined.

On the other hand, in a region where the scattering vector q is relatively large, it is a rod-like protein, and when the relationship between the scattering vector q and the center-of-gravity average radius Rc is q ² * Rc ² <1, that is, the thickness of the rod When the corresponding scattering intensity I (q) _thin has a relationship of Lπ / q with respect to the rod length L, ln (I (q) = K + (− ½ * Rc ² * q ² ). Therefore, Rc can be obtained from the slope of a straight line obtained by plotting with ln (I (q) and q ^{2. The} actual cross-sectional radius R can be obtained from R = Rc ^1/2 (references; H. Matsuoka et al., J. Colloid.Interface Sci., 118, 387 (1987)).

  Here, for example, UCH-L1 having a ubiquitin recycling function related to the onset of Parkinson's disease, normal UCH-L1 protein structure, Parkinson's disease risk amplification factor UCH-L1 (I93M substitution) and Parkinson's disease risk A protein was synthesized and purified from a human gene of UCH-L1 (S18Y substitute), which is a mitigating factor, using E. coli, and the structure was analyzed in deuterium. As a result, (1) UCH-L1 exists as a dimer in water. (2) The structure of the monomer that forms the dimer is different, and UCH-L1 (I93M substitution product), which is a Parkinson's disease risk amplification factor, is more elliptical than the normal type, whereas Parkinson's disease risk It was found that UCH-L1 (S18Y substitution product), which is a mitigating factor, has a low ellipticity and is a substantially spherical dimer. The globularity of the protein is also supported by the increase (increase in globularity) and decrease (increase in ellipticity) of the β-turn (folding structure) inside the protein by circular dichroism measurement.

  It was revealed for the first time that the hydrolytic activity, which is a ubiquitin recycling function, was higher in the order of I93M <normal type <S18Y, and was consistent with the globularity of the protein. If this method is used, a substantial structural change in water due to a gene mutation of the protein is revealed, and thus pathological prediction and diagnosis are possible.

  There is no direct in vitro comparison between the single molecule structure of normal microtubule-binding protein tau in water and the tau fiber structure (pathological state) found in brain disease. In the neutron small angle scattering method, tau molecules partially cross-linked by SS bonds form long filaments with a diameter of 36 mm, but when SS bonds are completely broken, tau spontaneously becomes spherical with a radius of 350 mm. It became clear to form a cluster. This facilitates the elucidation of the process from a normal state to a pathological state, and enables the development of a medicine that suppresses abnormal protein fibrosis that induces cell degeneration and cell death.

  In addition, if the target protein treated with the test substance or the target protein separated from the animal to which the test substance is administered is used as a sample and the form is measured by the neutron small angle scattering method as described above, the test substance becomes a pharmaceutical product. Can be screened as useful. Here, the target protein may be synthesized or separated from a living body, like the test protein.

  According to the present invention, the protein involved in the pathogenesis is extracted and purified directly from the living body, or when it is difficult to extract the protein directly such as the brain, the protein is synthesized against the sample synthesized and purified from the cloned gene. Diagnosis can be carried out by directly observing the protein structure (complex) according to the invention and comparing it with the normal type.

  In addition, for dimerization proteins such as UCH-L1, gene therapy can open the way to the possibility of expressing S18Y globular protein in I93M mutant Parkinson's disease patients and restoring enzyme function. It becomes a screening method for a drug that restores the shape of abnormal protein normally (spheroidization) and restores its function. In addition, it is possible to screen for inhibitors against abnormal protein association / aggregation such as tau.

  It is possible to compare various proteins that are candidates for pathological factors extracted from patients with diseases and normal proteins extracted from normal people, examine which protein forms have changed, and clarify the cause of the onset it can. Moreover, when a test protein cannot be extracted directly as in a brain disease, a diagnosis can be performed by synthesizing a protein from a gene extracted from a patient and comparing it with a normal protein. Furthermore, even in the case of a disease in which an abnormal macromolecule is observed, such as fibrosis due to a metabolic abnormality, even if it is a normal protein, the normal protein, regardless of whether the protein is directly extracted or synthesized from a gene By applying various treatments such as phosphorylation and oxidative stress and comparing the forms before and after loading, it is possible to determine what abnormal metabolic processes cause normal proteins to become polymerized and to clarify the cause of the onset be able to. In addition, based on these findings, we will determine the current treatment policy, develop gene therapy, develop new drug screening methods that suppress abnormal association and aggregation of proteins, and plan disease prevention measures. It becomes possible.

  In the present invention, the disease to be diagnosed or screened for a drug is a disease caused by a change in the shape or size of the protein, for example, familial Parkinson's disease associated with an abnormal gene of synuclein, which is a brain protein, Examples include idiopathic Parkinson's disease and familial Parkinson's disease that has a ubiquitin recycling function and is related to UCH-L1 gene abnormality. In addition, neurofibrillary tangle dementia called Taupathy includes general Alzheimer's disease, frontotemporal lobe dementia in which tau gene mutation is observed, and neurofibrillary dominant dementia in which no senile plaque is observed. On the other hand, when observed with an electron microscope, tau PHF (paired helical filament) having a repetitive structure with a period of 80 nm is observed in hereditary diseases, developmental abnormalities, and inflammatory diseases, but autosomal recessive In genetic diseases, metabolic diseases such as Niemann-Pick type C, degenerative diseases such as Hallervoden-Spatz syndrome, and autosomal dominant genetic diseases include muscular dystrophy and familial prion disease. There are also chronic diseases such as subacute sclerosing panencephalitis. In familial Pick's disease, abnormal tau aggregates, which are the main component of PHF (paired helical filament), are observed. Even in normal tau (four repeat tau), for example, in the case of progressive supranuclear palsy or cortical basal ganglia degeneration, aggregates are observed. Furthermore, examples of diseases in which polyglutamine accumulates include Huntington's chorea and Machado Joseph disease. The present invention is not limited to these brain diseases, but includes biological diseases such as viral diseases such as cancer and AIDS, infectious diseases, metabolic disorders, and congenital diseases, which are accompanied by cell death caused by various protein structural abnormalities. It can also be applied to diagnosis and treatment of protein structure abnormalities in various tissues and cells over a wide range of areas, and screening for new drugs.

Example 1
Diagnosis of Parkinson's disease by structural analysis of UCH-L1 gene mutant protein by neutron scattering method Normal human UCH-L1 cDNA and cDNA with I93M, S18Y, I93M + S18Y mutation added thereto were prepared. Each cDNA was subcloned into the expression vector pPROtetE233 for E. coli and introduced into E. coli strain DH5aPRO. The obtained transformant was cultured with shaking in a medium growth medium at 37 ° C. until the OD was about 0.5 (about 2 hours), and isopropyl-β-D-thiogalactoside was brought to a final concentration of 0.1 mM. After adding (IPTG), the cells were further cultured for 6 hours. The cells were collected by centrifugation (5000 × g, 20 minutes, room temperature) and suspended in 20 mM HEPES, pH 7.8. Bacteria were lysed by ultrasonic disruption, and UCH-L1 was purified using Co ²⁺ -Sepharose (TALON purification kit, Clontech) according to the instructions. This was lyophilized and stored at −80 ° C. (references; K. Nishikawa, et. All., BBRC, 304, 176-183 (2003)).

Neutrons dissolved in a buffer solution using heavy water (finally 50 mM HEPES pH 7.8, 5 mM dithiothreitol (DTT)) to a concentration of 0.86 mg / mL and installed at the High Energy Accelerator Research Organization Neutron scattering measurement was performed at room temperature using a scattering device (WINK, 0.03 <q (Å ⁻¹ ) <0.15). Here, q is a momentum change expressed as q = (4π / λ) sin θ, where the Bragg angle is θ. The transmittance of the solvent and the UCH-L1 solution was measured, the background was subtracted, and each scattering intensity obtained by standardization was determined. The value obtained by subtracting the scattering intensity of the solvent from the scattering intensity from the UCH-L1 solution was used as the scattering intensity of neutrons scattered from the protein.

  Next, the form of the protein in the aqueous solution is analyzed. The protein morphology was determined so that the theoretical neutron scattering curve predicted from the protein structure in water and the experimentally obtained UCH-L1 neutron scattering curve most closely matched. More specifically, although the crystal structure of UCH-L1 is not yet known, UCH-L1 has a hydrolase activity (50% amino acid sequence matches) and is widely distributed in the living body, like UCH-L1. Analyzed by SWISS-MODEL from crystal structure analysis data of L3 (reference document: Schwede, T. et. Al., Nucleic Acids Res. 31, 3381-3385 (2003)., Guex, N. et al., Electrophoresis 18, 2714-2723 (1997)., Peitsch, MC, Bio / Technology 13, 658-660 (1995).), The crystal structure of UCH-L1 was calculated (FIG. 3). Next, when the rotation radius of the protein is calculated from the crystal structure information (reference: Svergun, DI et al., Proc. Matl. Acad. Sci. USA, 95, 2267-227) (1998). Svergun, D .; I. et al. , J .; Appl. Cryst. 28, 768-77) (1995). ), The center of gravity rotation radius Rg is 16.5 mm (the actual rotation radius is

Was assumed to be a globular protein. From the calculated globular protein radius, a neutron scattering curve of the monomer of UCH-L1 in heavy water can be theoretically obtained by the following equation (1). The dimer can also be determined from the distance between the centers of the two molecules.

FIG. 4 shows the calculated theoretical curve from the monomer and dimer of the globular protein UCH-L1, and the actual measurement results of the normal type. It was clearly close to the dimer, not the monomer, but was found to be less spherical. Therefore, if the number of amino acids is constant, the volume of the protein does not change so much even if it is deformed. Therefore, the shape of the protein is gradually changed to a rotating disk (minor axis; a, major axis; b, (= c)). Alternatively, it was deformed as a spindle (minor axis; a, (= b), major axis; c), and monomers and dimers were predicted as these forms. All these proteins are divided into 5 cm cubes, and the scattering length density difference between the divided cubes is extremely small compared to the difference between hydrogen atoms (H) and deuterium (D). The theoretical neutron scattering curve from the aqueous solution was obtained by the following equation (2) by calculating the correlation between the sites. FIG. 5a shows a conceptual diagram in the case of obtaining all the correlations between 5 cubic cubes of monomers or dimers. FIG. 5b shows the relationship between the scattering vector q and the scattering intensity I (q) when the minor axis, a, is gradually changed in a monomer rotating disk (minor axis; a, major axis; b, (= c)). Show the relationship. FIG. 5c shows a scattering vector q and a scattering intensity I (q when the minor axis, a, is gradually changed in a rotating disk body (minor axis; a, major axis; b, (= c)) in the case of a dimer. ). Furthermore, FIG. 5d shows that even if the dimer is a spindle (minor axis; a, (= b), major axis; c), the scattering vector q and the scattering intensity I (q ). Only in the rotating disk body dimer of FIG. 5b, characteristic scattering intensity attenuation is observed in the range of 0.1 <q (Å ⁻¹ ) <0.15, and the UCH-L1 scattering curve of FIG. Is the closest to the rotating disk.

  Next, fitting was performed using the following equation (3) so that the difference between the experimental value and the theoretical value was minimized.

Here, m is a scaling factor, n is a background factor, I _e (q) is a scattering intensity actually obtained by neutron scattering measurement, and I _c (q) is a scattering intensity obtained theoretically. The form of the protein with the smallest R was taken as the structure in the aqueous solution of UCH-L1. Since the spindle did not match the actual measurement curve unless the spindle had a very long major axis, a rotating disk was used. As shown in FIG. 6, the normal UCH-L1, I93M, S18Y, I93M + S18Y are all dimers, but the monomers forming the dimer are normal types [minor axis, 29 mm; major axis, 52 mm ( 6a)], I93M [minor axis, 20 mm; major axis, 62 mm (FIG. 6b)], S18Y [minor axis, 43 mm; major axis, 43 mm (FIG. 6c)], I93M + S18Y [minor axis, 31 mm; major axis, 50 mm (FIG. 6d). )]Met. That is, it was found that the spherical nature of the monomer in water differs depending on the amino acid substitution. It is known that the change in sphericity of UCH-L1 due to amino acid substitution correlates well with the β-turn (peptide chain folding) content in the secondary structure of the protein. FIG. 7 shows how the α-helix, β-sheet, β-turn, and random coil component were changed by amino acid substitution from the circular dichroism measurement results. Compared with the normal type, I93M substitution showed an increase in β-sheet and a decrease in β-turn. On the other hand, compared to the normal type, an increase in β-turn was observed by S18Y substitution. Furthermore, even when compared with the I93M substitution product, an increase in β-turn was observed by the S18Y substitution. That is, as shown in Table 5, the change in sphericity of UCH-L1 by amino acid substitution obtained from neutron scattering experiments and the change in sphericity of UCH-L1 with β-turn as an index by circular dichroism measurement are good. It was confirmed that they matched.

From these results and the hydrolytic activity that is the function of UCH-L1, that is, the intracellular recovery function of ubiquitin, as summarized in FIG.
(1) UCH-L1 forms a dimer in water.
(2) The sphericity of the monomer of UCH-L1 that forms the dimer is changed by amino acid substitution, and the I93M substitution has a more spherical shape, whereas the S18Y substitution has more sphericity.
In the I93M substitution product modified from (3), the hydrolytic activity decreases and becomes a Parkinson's disease risk amplification factor.
(4) In the S18Y substitution product having higher globularity, the hydrolytic activity increases, and becomes a Parkinson's disease risk reduction factor to suppress the onset.

  The above became clear. Thus, by using the neutron scattering method, it is not only possible to clarify the form of the protein in water in line with the living body, which was difficult to predict from the conventional crystal structure analysis, but also the protein. It was found that Parkinson's disease can be diagnosed by comparing the form of with normal protein.

Example 2
Diagnosis of Alzheimer's disease by structural analysis of microtubule-binding protein tau by neutron scattering method cDNA of human Tau 40 protein (441 amino acids) was subcloned into expression vector pRK172 for E. coli and introduced into E. coli strain BL21 (DE3) pLysS. The obtained transformant was cultured with shaking in a circle growth medium at 37 ° C. until the OD was about 0.5 (about 2 hours). After adding IPTG to a final concentration of 0.1 mM, Cultured for 6 hours. The cells were collected by centrifugation (5000 × g, 20 minutes, room temperature) and suspended in PC buffer (20 mM MES-NaOH, pH 6.8, 0.5 mM Mg Acetate, 1 mM EGTA, 2 mM PMSF). The cells were dissolved by ultrasonic disruption, 1/150 times the amount of 2-mercaptoethanol and NaCl at a final concentration of 0.8 mM were added, followed by treatment at 95 ° C. for 15 minutes. After cooling, the supernatant was collected by centrifugation (20000 × g, 30 minutes, 4 ° C.) and dialyzed against PC buffer overnight. PC11 resin equilibrated with PC buffer and the dialyzed protein solution were mixed and stirred at 4 ° C. for 5 hours or more. After thoroughly washing the resin with a PC buffer containing 0.1 mM NaCl, the Tau 40 protein fraction was eluted with a PC buffer containing 0.5 mM NaCl. The Tau 40 protein fraction was dialyzed overnight against MilliQ water, further replaced with 50 mM Tris-HCl, 5 mM EDTA, 10 mM DTT, pH 7 buffer, lyophilized, and subjected to neutron scattering experiments. In addition, once an SS bond is formed between tau molecules by air oxidation in the tau purification process, it cannot be reduced or cleaved easily. Therefore, in order to eliminate the polymerization phenomenon due to SS bond, 20 mM DTT is further added. In addition, neutron scattering measurement was similarly performed on tau heated at 70 ° C. to reduce and cut SS bonds. The polymerized tau is produced by the Japan Atomic Energy Research Institute neutron scattering system (SANS-J; 0.0013 <q (Å ⁻¹ ) <0.014) and the High Energy Accelerator Research Organization neutron scattering apparatus (SWAN; 0 0.009 <q (Å ⁻¹ ) <0.99). In addition, the low concentration tau purified by reducing and cleaving the SS bond was measured by focusing 40 neutrons into the neutron scattering apparatus of the Japan Atomic Energy Research Institute to collect neutrons.

The results are shown in FIG. The tau before reducing and cleaving the SS bond is dissolved in water by forming a random coil because the slope of the straight line is proportional to q ⁻² , as seen in q <˜0.008Å− ^1. Cross-sectional analysis in the range of 0.009 <q (Å ⁻¹ ) <0.025 (reference: H. Matsuoka et al., J. Colloid. Interface Sci., 118, 387 (1987)) (inset in FIG. 5), it was found that the diameter was 36 mm. On the other hand, after the reduction and cleavage of the SS bond, the scattering curve is completely different from that before the treatment, and as q decreases, the scattering intensity does not increase, and as q increases, the scattering intensity decreases to q ⁻⁴ . From the proportionality, it was found that the particles were spherical. According to the Gunier method (reference: A. Gunier, et.al., “Small-angle Scattering of X-rays”, Wiley, New York (1955)), the turning radius of the particle is 350 mm, and several tens of tau It was found that the molecules assembled by self-assembly and formed spherical clusters. Even in normal tau, aggregates have been found in various brain diseases. Therefore, according to this analysis, normal tau molecules bind to other proteins in aqueous solution or form aggregates due to weak intermolecular interactions, but when oxidative stress is applied to cells, It was found that SS bonds were introduced and fibrillated. The evaluation system using the neutron scattering method can reproduce the abnormal protein conformation phenomenon occurring in the brain cells in vitro, and can screen for new drugs that inhibit or suppress this phenomenon.

Claims (6)

  A method for diagnosing a disease caused by a change in the shape of a test protein, comprising irradiating a test protein dissolved or dispersed in heavy water with cold neutrons and measuring the form of the test protein from the scattering of the cold neutrons .   The diagnostic method according to claim 1, wherein the morphological change of the test protein is a change in the shape and / or size of the test protein.   The diagnostic method according to claim 1 or 2, wherein the test protein is synthesized using a gene derived from a living organism or separated from a living organism.   A pharmaceutical screening method using a target protein treated with a test substance or a target protein separated from an animal administered with the test substance, wherein the target protein dissolved or dispersed in heavy water is irradiated with cold neutrons, A method for screening a therapeutic agent for a disease caused by a change in the shape of a target protein, comprising measuring the shape of the target protein from the scattering of the cold neutrons.   The screening method according to claim 4, wherein the morphological change of the target protein is a change in the shape and / or size of the target protein.   The screening method according to claim 4 or 5, wherein the target protein is synthesized using a gene derived from a living body or separated from a living body.

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