CN101724057A

CN101724057A - Application of amyloid precursor protein N-end structure in transport and metabolism process

Info

Publication number: CN101724057A
Application number: CN200810201708A
Authority: CN
Inventors: 景乃禾; 陈永峰; 沈承勇
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 2008-10-24
Filing date: 2008-10-24
Publication date: 2010-06-09

Abstract

The invention relates to application of an amyloid precursor protein N-end structure in the transport and metabolism process and discloses a protein fragment separated from amyloid precursor protein, wherein the protein fragment can be used for preparing or screening a substance which specifically acts on the protein fragment. The invention also discloses a method for screening a potential substance inhibiting neurodegenerative diseases by inhibiting the transportation or metabolism of the amyloid precursor protein, comprising the step of selecting a substance specifically acting on a target spot by taking an amyloid precursor protein exon 2 area as the target spot for screening. The invention provides a new path for screening medicaments for preventing and treating the neurodegenerative diseases.

Description

The effect of amyloid precursor protein N-end structure in its transportation and metabolic process

Technical field

The invention belongs to biological technical field, more specifically, the present invention relates to the effect of amyloid precursor protein N-end structure in its transportation and metabolic process.

Background technology

Senile dementia be in, modal a kind of nerve degenerative diseases in the elderly population, Clinical symptoms comprises the decline of memory, the forfeiture of cognitive ability, serious dystropy also finally causes death.AD patient's brain sheet shows three kinds of unusual pathological characters: a large amount of minimizings of senile plaque, neurofibrillary tangles and neurocyte.The main ingredient of senile plaque be the amyloid beta that contains 39～43 amino-acid residues ( β- aMyloidprotein, A β), it mainly contains two kinds of forms: A β 40 and A β 42, is made up of 40 and 42 amino acid respectively, and wherein A β 42 mainly is distributed in patient's AD brain, is cytotoxic form.With A β neurotoxicity is that the A beta hypothesis at center has become the well accepted AD pathogenesis of present academia.

A β be by amyloid precursor protein ( AMyloid pRecursor pRotein is APP) through the Secretases hydrolysis.App gene is positioned on No. 21 karyomit(e), has 18 exons, is processed to form different coded product APP770, APP751 and APP695 etc. through shearing.APP695 (lacking the 7th, the 8 two exon) specifically expressing is in brain, and especially high expression level in neuronal cell is the closest with the generation relation of A β.

APP albumen single is striden film, belongs to I type transmembrane protein, and it comprises secretion and two processes of endocytosis at intracellular transport pathway.After APP synthesizes in rrna, at first by intracellular transport pathway, pass through endoplasmic reticulum successively, golgi body, betransported vesicle and be transported to cell surface, under the guidance of the inner endocytosis signal of its born of the same parents, brought back in the cell by the endocytosis vesicle then by intracellular endocytic pathway, enter the endocytosis body, in the organoids such as lysosome.In said process, the various Secretases that APP can be located on the different organoids cuts, and produces different meta-bolitess.The endocellular metabolism of APP mainly contains two kinds of different approach, according to finally whether producing A β, can be divided into non-amyloid approach and amyloid approach.((β-secretase) has mediated this two processes so be otherwise known as alpha-secretase enzymes metabolism approach and beta-secretase pathways metabolism respectively for α-secretase) and beta-secretase because the alpha-secretase enzyme.

As shown in Figure 1, under the normal physiological situation, APP in cell mainly at surface of cell membrane by non-amyloid pathways metabolism by metabolism, APP is earlier under the effect of alpha-secretase enzyme, generation contains secretion APP (the secretory APP of part A β sequence, sAPP α) and comprise 83 amino acid whose C-terminal transmembrane segments (APPC-terminal fragment, C83), this C-terminal fragment is then further degraded in cell.Metalloenzyme family member ADAM9, ADAM10, ADAM17 has alpha-secretase activity.The action site of alpha-secretase enzyme is positioned at A β sequence inside, can destroy the complete structure of A β after enzyme is cut, and therefore can not generate A β.And in the beta-secretase pathways metabolism, APP can locate by the beta-secretase effect at secretory vesicle or endocytosis body, lysosome etc., generate secretion property sAPP β and the C-terminal fragment (C99) that contains A β complete structure, beta-secretase (β-secretase) further on film with the hydrolysis of C99 segment, generate A β and APP intracellular region (APPintracellular domain, AICD) segment.Beta-secretase claims BACE (beta-site APP-cleavingenzyme) again, is a special member of aspartate protease family.Gamma-secretase is a mixture of being made up of four kinds of protein, comprise presenilin albumen 1 (Presenlin 1, PS1), Nicastrin, Aph-1 and Pen-2, what wherein bring into play katalysis is the aspartic acid hydrolytic enzyme activities that asparagicacid residue provided that PS 1 strides membrane portions.Its action site on APP is not single-minded, and the A β of generation mainly contains 42 two kinds of A β 40 and A β, and wherein A β 42 hydrophobicitys are stronger, deleterious A beta oligomers of easier formation and deposition under physiological condition.

In work in the past, the inventor has been cloned into a kind of new montage product A PP639 (APP-Δ E2) of people's app gene.Compare a large amount of APP695 albumen that exist in the brain, APP-Δ E2 lacks 56 amino acid (E2 district) of second exons coding of N end, referring to ZL03129585.1.

Although those skilled in the art have made number of research projects for APP albumen in the prior art, yet each section is clear not enough for the understanding of APP self metabolism adjusting aspect on the APP albumen.Therefore, be necessary each section on the APP albumen is carried out finer research, to find suitable drug target, the screening that prevents and treats medicine for nerve degenerative diseases provides effective way.

Summary of the invention

The object of the present invention is to provide a kind ofly for the useful drug target of screening nerve degenerative diseases control medicine, described target spot is positioned at the N end of amyloid precursor protein (APP).

Another object of the present invention is to provide a kind of method based on described drug target screening of medicaments.

In a first aspect of the present invention, a kind of isolating amyloid precursor protein fragment is provided, the aminoacid sequence of described protein fragments is shown in 21-76 position among the SEQ ID NO:1 (56aa).

In a second aspect of the present invention, the purposes of described protein fragments is provided, be used to prepare or screen the material of specific effect in this protein fragments.

In another preference, described material is selected from but is not limited to: peptide, pdef polypeptide, plan peptide, non-peptide compound, carbohydrate, fat, antibody or antibody fragment, part, organic molecule, inorganic molecules and nucleotide sequence.

In another preference, described material is selected from: specificity is in conjunction with the antibody of this protein fragments, and specificity is in conjunction with the part of this protein fragments, and specific effect is in the micromolecular compound of this protein fragments (activity that preferably blocks this protein fragments).

In a third aspect of the present invention, a kind of method of screening the transportation that suppresses amyloid precursor protein or metabolism and then suppressing the potential material of nerve degenerative diseases is provided, described method comprises:

With the amyloid precursor protein that contains the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1 or fragment target spot as screening, select specific effect in this target spot material of (as suppressing, block or reducing the segmental activity of albumen on this target spot), described material is transportation or the metabolic potential material that suppresses amyloid precursor protein.

In another preference, described method comprises:

(1) provide a kind of amyloid precursor protein or fragment, described albumen or fragment contain the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1;

(2) with the described amyloid precursor protein fragment of candidate substances treatment step (1);

(3) select specific effect candidate substances in the aminoacid sequence site shown in the 21-76 position among the SEQ ID NO:1 in described albumen or fragment, described material is transportation or the metabolic potential material that suppresses amyloid precursor protein.

In another preference, described candidate substances is selected from (but being not limited to): peptide, pdef polypeptide, plan peptide, non-peptide compound, carbohydrate, fat, antibody or antibody fragment, part, organic molecule, inorganic molecules and nucleotide sequence.

In another preference, described candidate substances is compound (a preferred micromolecular compound), antibody or part.

In another preference, described method comprises:

(1a) provide a kind of reference protein fragment, described reference protein fragment does not contain the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1, and other aminoacid sequence is identical with the amyloid precursor protein or the fragment of (1);

(2) use candidate substances treatment step (1) and (1a) described albumen or fragment respectively;

(3) select amyloid precursor protein or the fragment that acts on step (1), and do not act on the segmental material of reference protein of step (1a), described material is transportation or the metabolic potential material that suppresses amyloid precursor protein.

In another preference, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, further to select from candidate substances and to determine for the transportation or the useful material of metabolism that suppress amyloid precursor protein.

In another preference, described method also comprises:

(4) the potential mass treatment cell that obtains with step (3), described cell expressing amyloid precursor protein or fragment (for example expressing APP695 albumen), described albumen or fragment contain the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1;

(5) observe the interior amyloid precursor protein of cell of step (4) or the common location situation of fragment and organoids such as endoplasmic reticulum, golgi body, transport vesicle or endocytosis body, if not having common location, then described potential material basically is transportation or the metabolic material that suppresses amyloid precursor protein.

In another preference, described method also comprises:

The potential mass treatment cell that (4 ') obtains with step (3), described cell expressing amyloid precursor protein or fragment, described albumen or fragment contain the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1;

The amount of amyloid precursor protein C end fragment (CTF) in the cell of (5 ') determination step (4 '), and with control group relatively; Wherein said control group is expression amyloid precursor protein or the segmental cell without the potential mass treatment of step (3) acquisition;

If the amount of amyloid precursor protein C end fragment is lower than statistically in the cell of described potential mass treatment (as low 20%; Preferably hang down 50%; More preferably low 100% or lower) amount of amyloid precursor protein C end fragment in the cellular control unit, then described potential material is transportation or the metabolic material that suppresses amyloid precursor protein.

In another preference, described amyloid precursor protein C end fragment comprises: C99, C83, amyloid precursor protein intracellular region (AICD), A β 40, A β 42.

In another preference, described method also comprises:

The potential mass treatment cell that (4 ") obtain with step (3), described cell expressing amyloid precursor protein or fragment, described albumen or fragment contain the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1;

Whether form patch structure in the cell of (5 ") determination step (4 "), if form patch structure, then described potential material is transportation or the metabolic material that suppresses amyloid precursor protein.

In a fourth aspect of the present invention, provide a kind of transportation or metabolic material of the inhibition amyloid precursor protein that obtains by described method.

In another preference, described material is an antibody.

In a fifth aspect of the present invention, a kind of antibody of anti-amyloid precursor protein is provided, described antibodies specific ground is in conjunction with described protein fragments.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Description of drawings

Two kinds of metabolic waies of Fig. 1 .APP comprise alpha-secretase enzymes metabolism mode and beta-secretase metabolic way.

The disappearance of Fig. 2 .APP albumen n end E2 causes the metabolic downward modulation of APP.

The CTF that A.APP-Δ E2 metabolism produces is less than APP695 and APP-Δ E3, and swimming lane 1-4 is an alpha-secretase enzymes metabolism process, and swimming lane 5-8 is the beta-secretase metabolic process, and CTF comprises C99myc, C83myc and AICDmyc.

The A β amount that B.APP-Δ E2 metabolism produces is less than APP695 and APP-Δ E3.

The C.APP structural representation.

The disappearance of Fig. 3 .APP albumen n end E2 makes that the alpha-secretase enzymes metabolism process of APP and beta-secretase metabolic process are all reduced.

The α of A.APP-Δ E2-and the product C TF and the sAPP of beta-secretase metabolic process all be less than APP695, swimming lane 1-6 is an alpha-secretase enzymes metabolism process, swimming lane 7-12 is the beta-secretase metabolic process, the identification sAPP α of wo-2 antibodies specific, the sAPP β that the identification APP695 of sAPP β antibodies specific produces can not discern the sAPP β that APPsw produces.

B.APP-Δ E2 can not locate altogether with BACE, and APP695 can locate altogether with BACE, and white portion is for being total to localized APP and BACE.

The disappearance of Fig. 4 .APP albumen n end E2 makes the Subcellular Localization of APP be affected.

A.APP-Δ E2 does not have the albumen of mature form to exist, and APP695 and APP-Δ E3 have the albumen after the corresponding modification, and myc antibody and wo-2 antibody all can be discerned the APP-myc of total length.

B.APP-Δ E2 does not locate altogether with different organoid marks (Marker), and APP695 and these marks can be located altogether.Wherein, LMAN1 is the mark that endoplasmic reticulum arrives the transport vesicle of golgi body, and GM130 is the mark of golgi body, and EEA1 is the Marker of early stage endocytosis body, and M6PR is the mark of endocytosis body in late period.

The APP metabolism of the inner Cys sudden change in Fig. 5 .APP albumen n end E2 district is consistent with APP-Δ E2 with the location situation.

A.C is similar to the CTF and the APP-Δ E2 of the APP-myc metabolism generation of G sudden change, all obviously is less than APP695, and swimming lane 1-6 is an alpha-secretase enzymes metabolism process, and swimming lane 7-12 is the beta-secretase metabolic process.

B.C is to the APP-EYFP and the similar accumulative patch structure that forms in cell of APP-Δ E2 of G sudden change.

The formed special patch structure of Fig. 6 .APP-Δ E2 is not an aggregate.

The mark of A.APP-Δ E2 and aggregate is not all located altogether, and Vimentin, γ-tubulin, Ubiquitin are the marks (Marker) of aggregate.

B.APP-Δ E2 is the same with APP695, in the component of gentle washing agent solubility and non-solubility distribution is arranged all, and s is a soluble component, and i is the non-solubility component.

Embodiment

The inventor is through extensive and deep research, the N end structure that discloses amyloid precursor protein (APP) has first been brought into play vital role in the transportation of amyloid precursor protein and metabolic process, destroying the proteic N end structure of APP can be so that transportation and the metabolic disturbance of APP in the cell, and the generation of toxic product significantly reduces.New discovery based on the inventor, can be with the target spot of the proteic N end structure of APP as drug screening, thereby screening can suppress APP albumen normal transport and metabolic material in cell by suppressing this target spot, and described material is the potential material that can prevent and treat nerve degenerative diseases.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute "; " mainly by ... constitute ", " basically by ... constitute " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".

As used herein, " specific effect is in the amyloid precursor protein fragment " is meant that a kind of material can interact with 2 zones of intron in the amyloid precursor protein (preferably having the site of aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1), thereby suppresses, blocks or reduce the activity or the function in this zone.Transportation or the metabolism in cell exerts an influence to amyloid precursor protein for the inhibition of described regional activity or function, retardance or downward modulation.

As used herein, " specificity " of antibody is meant that antibody capable is incorporated into intron 2 zones of amyloid precursor protein (preferably having the site of aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1), thereby suppresses, blocks or reduce the activity or the function in this zone.

As used herein, unless otherwise indicated, " amyloid precursor protein or fragment " is the amyloid precursor protein of total length, the amyloid precursor protein of various shear-forms, the general name of amyloid precursor protein extracellular region protein, amyloid precursor protein N-end structure etc.

Herein, unless otherwise indicated, described nerve degenerative diseases is meant that a class produces the relevant nerve degenerative diseases of cytotoxicity fragment (as A β 42) with the amyloid precursor protein metabolism, as senile dementia (being called Alzheimer's disease again).

Drug target and uses thereof

Amyloid precursor protein (APP) major part is positioned at the extracellular, and the N end contains 17 amino acid whose signal peptides, and part is very short APP PROTEIN C end in the born of the same parents.Because the C end has comprised A β sequence, is the zone of action of various Secretasess.Up to the present also do not obtain the complete crystalline structure of APP albumen, the analysis of three-dimensional structure mainly is confined to APP extracellular region territory.The proteic aminoacid sequence of a kind of APP for example can (APP695, the GenBank accession number: NP_958817) substantially the same, APP695 is specific expressed in brain, and be closely related with the generation of A β with the sequence shown in the SEQ ID NO:1.

The APP albumen n end sequence of different genera is very conservative, and amino acid identity is up to 36-84%.The outer N end regions of APP albumen born of the same parents contains the folding and β spiral of nine α, constitutes a globosity closely.The N end of APP is rich in halfcystine, forms three disulfide linkage, and whole APP protein extracellular territory is tightened together.The proteic N end of APP has a heparin-binding region, and known ectogenic heparin can influence the secretion of A β.The N end regions has more basic aminoacids, and the positive charge that has can interosculate with the glycosaminoglycan that brain is rich in; Contiguous hydrophobic region may have ligand binding capacity, maybe can form self dimer; The somatomedin that the structure in APP albumen n end zone and some are rich in halfcystine is similar.

The inventor has proved that through a large amount of work the integrity of APP albumen n end structure has important effect for proteic normal transport of APP and metabolism.The inventor has made up albumen (the APP-Δ E2 of APP albumen n end exon 2 (E2) disappearance, this disappearance makes that the proteic N end structure of APP is destroyed), find that APP-Δ E2 is starkly lower than APP695 or some other APP shear-form at intracellular metaboilic level, this metabolic disturbance mainly occurs in the process of alpha-secretase enzyme, beta-secretase effect, and the effect of gamma-secretase is also unaffected.And APP-Δ E2 also has different with APP695 or some other APP shear-form in intracellular Subcellular Localization situation.The Cys rite-directed mutagenesis that the E2 district is inner can obtain the similar result with APP-Δ E2.The above results has also pointed out the disappearance in E2 district to cause that the false folding of APP causes APP Proteometabolism and localized unusual.

And the inventor also finds, after the proteic N end structure of APP is destroyed, mainly exists with accumulative patch form in cell.Cell has multiple coping mechanism for the albumen of false folding, wherein a kind of is to make the albumen of false folding form the structure of aggregate (Aggresome) around centrosome by fibrinous effect, but this special structure that APP-Δ E2 forms not is an aggregate structure.

Based on the inventor's above-mentioned new discovery, the invention provides a kind of isolating amyloid precursor protein fragment, described protein fragments is the protein fragments that separates from the exon 2 zone of amyloid precursor protein.Preferably, the aminoacid sequence of described protein fragments is shown in 21-76 position among the SEQ ID NO:1 (56aa).Described protein fragments is used to screen the material that specific effect can take place with it, and described material for example can be selected from (but being not limited to): peptide, pdef polypeptide, plan peptide, non-peptide compound, carbohydrate, fat, antibody or antibody fragment, part, organic molecule, inorganic molecules and nucleotide sequence.

As optimal way of the present invention, can be with described protein fragments as a kind of antigen, the preparation specificity is in conjunction with this antigenic antibody.Also can obtain the antibody of some anti-amyloid precursor proteins as antigen based on the amyloid precursor protein of total length, yet these antibody may be incorporated into epitopes different on the amyloid precursor protein, will therefrom filter out the antibody that specificity is incorporated into amyloid precursor protein exon 2 zone needs a large amount of work, and screening efficiency is not high.Therefore, directly carry out the preparation of antibody as antigen, can reduce the workload of antibody screening widely with protein fragments of the present invention.

In addition, also can prepare a series of antibody as antigen, but select the antibody of specific effect by Ag-Ab calmodulin binding domain CaM analytical technology again in the exon 2 zone based on total length or the amyloid precursor protein shear-form that contains exon 2.The epitope scanning technique that a kind of available analytical technology for example is a monoclonal antibody, but this technology referenced patent application number for example: 200710173305.2.

In case separate the sequence that has obtained described protein fragments, just can obtain this protein fragments in large quantity with recombination method.This normally is cloned into carrier with its encoding gene, changes cell again over to, separates obtaining then from the host cell after the propagation by ordinary method.In addition, for short albumen, also can adopt the method for synthetic (as synthetic by Peptide synthesizer) to synthesize relevant sequence, the method for synthetic can obtain needed albumen easy and apace.The present invention also provides the purposes of described protein fragments, and it is used to prepare the antibody of specificity in conjunction with amyloid precursor protein as the fragment of a kind of effective antigens of amyloid precursor protein, comprises monoclonal antibody or polyclonal antibody.

Described antibody can be prepared by the known various technology of those skilled in that art.For example, the protein fragments of the present invention of purifying can be applied to animal to induce the generation of polyclonal antibody.Similarly, the cell of expressing described protein fragments can be used to immune animal and produces antibody.Described antibody also can be monoclonal antibody, and it can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981).The available protein fragments immune animal of the present invention of the production of polyclonal antibody, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

As optimal way of the present invention, with the amyloid precursor protein that contains described protein fragments or its shear-form as screening model, but the screening specific effect is in the material of described protein fragments, for example micromolecular compound.

Screening method

The exon 2 zone of cicada amyloid precursor protein brought into play vital role in the transportation of amyloid precursor protein and metabolic process after, can screen the exon 2 zone of specific effect based on this new discovery, and then influence transportation and the metabolic material of amyloid precursor protein in cell in amyloid precursor protein.By influencing transportation and the metabolism of amyloid precursor protein in cell, the pathways metabolism of downward modulation α-Dian Fenmei and beta-amylase can reduce the generation of toxicity a (as A β 42) in the cell effectively, alleviates nerve degenerative diseases effectively.

Therefore, the invention provides a kind of transportation of inhibition amyloid precursor protein or method of metabolic potential material of screening, described method comprises:

With the amyloid precursor protein in the exon 2 zone (the preferably aminoacid sequence shown in 21-76 position among the SEQ ID NO:1) of containing amyloid precursor protein or fragment target spot as screening, select specific effect in this target spot material of (as suppressing, block or reducing the segmental activity of albumen on this target spot), described material is transportation or the metabolic potential material that suppresses amyloid precursor protein.

As target spot, screening the method that acts on this regional material is that those skilled in the art are known with zone specific on the albumen, and these methods all can be used for the present invention.Described candidate substances can be selected from: peptide, pdef polypeptide, plan peptide, non-peptide compound, carbohydrate, fat, antibody or antibody fragment, part, organic molecule, inorganic molecules and nucleotide sequence etc.According to the kind of material to be screened, how those skilled in the art clear selects the screening method that is suitable for if being.

As optimal way of the present invention, described method comprises: (1) provides a kind of amyloid precursor protein or fragment, and described albumen or fragment contain the exon 2 zone of amyloid precursor protein; (2) with the described amyloid precursor protein fragment of candidate substances treatment step (1); (3) select the candidate substances of specific effect in described exon 2 zone, described material is transportation or the metabolic potential material that suppresses amyloid precursor protein.

As a kind of preferred mode, can 1. contain the amyloid precursor protein or the fragment in exon 2 zone and 2. not contain exon 2 zone and other regional with 1. identical amyloid precursor protein or fragment by test simultaneously; But judge specificity in conjunction with the former (1.) and debond in the material of the latter's (2.), also promptly this material acts on amyloid precursor protein exon 2 zone.After getting this scheme of cicada, concrete implementation method is easily for those skilled in the art.

Under a kind of filtering mode, carry out drug screening by simulating described amyloid precursor protein or the segmental three-dimensional structure that contains the exon 2 zone of amyloid precursor protein.A large amount of proteinic three-dimensional structures are resolved and are got by X ray crystalline diffraction technology and multi-dimensional nmr technology such as (NMR) at present, for the molecular recognition of studying the protein-ligand mixture provides favourable condition.The N end of amyloid precursor protein is positioned at outside the born of the same parents, can screen by the three-dimensional structure of simulating its extracellular region territory.

Many alternative screening storehouses (as micromolecular compound screening storehouse) have been arranged at present, made high flux screening become possibility.Can be by the area of computer aided medicinal design, by analyzing drug targets, it is the textural property of protein molecular functional site, design specific chemical small molecules, the each several part molecular structure of its molecule and physico-chemical property and target are complementary, with the purpose that reaches combination with it and play a role, these technology all are that those skilled in the art are known.

Through large-scale drug screening, can obtain the potential material of a class specific effect in amyloid precursor protein exon 2 zone.The potential material that these preliminary screening go out can constitute a screening storehouse, so that people finally can be on the basis of further checking, therefrom filtering out can be for the expression of regulating FSTL1 and active real useful material.

As optimal way of the present invention, also need the potential material that obtains is carried out further cell experiment and/or animal experiment, from these materials, further to select and to determine for the transportation or the useful material of metabolism that suppress amyloid precursor protein.

On cell levels, can carry out proof test by the mode that makes up cell model.The structure of relevant cell model is well known in the art.For example, can make it stably to express by amyloid precursor protein or segmental ceneme (as expression vector) are transferred in the host cell, transportation and metabolism amyloid precursor protein or fragment.The selection of ceneme and host cell is a technology well known in the art.Described host cell for example is (but being not limited to): HEK293.Certainly, also can be with endogenous expression amyloid precursor protein or segmental cell model as screening, described cell for example is (but being not limited to): the SK-N-SH cell.

As optimal way of the present invention, described method comprises: with described potential mass treatment cell, described cell expressing amyloid precursor protein or fragment, described albumen or fragment contain amyloid precursor protein exon 2 zone; Observe the common location situation of the interior amyloid precursor protein of this cell or fragment and organoids such as endocytoplasmic reticulum, golgi body, transport vesicle or endocytosis body, if not having common location, then described potential material basically is transportation or the metabolic material that suppresses amyloid precursor protein.A kind of technology of analyzing the common location situation of above-mentioned albumen and organoid is an immunofluorescence technique, and this is the known technology of those skilled in the art.The marker protein of corresponding organoid is that those skilled in the art are known.

As optimal way of the present invention, can determine after candidate substances is handled amyloid precursor protein or segmental metabotic change situation in the cell from alpha-secretase enzymes metabolism approach, beta-secretase pathways metabolism in the cell.Preferably, described method comprises: with described potential mass treatment cell, described cell expressing amyloid precursor protein or fragment, described albumen or fragment contain amyloid precursor protein exon 2 zone; Measure the amount of interior amyloid precursor protein C end fragment (CTF comprises C99, C83, amyloid precursor protein intracellular region (AICD), A β 40, A β 42 etc.) of this cell or secretion property APP (sAPP), and compare with control group; Wherein said control group is expression amyloid precursor protein or the segmental cell without the potential mass treatment of step (3) acquisition; If the amount of amyloid precursor protein C end fragment or secretion property APP is lower than the amount of amyloid precursor protein C end fragment in the cellular control unit or secretion property APP statistically in the cell of described potential mass treatment, then described potential material is transportation or the metabolic material that suppresses amyloid precursor protein.For example can adopt western blotting technology such as (as the Western traces) to come qualitative, above-mentioned each segmental content quantitatively or in the sxemiquantitative ground analysis of cells, this is a technology well known in the art.

As optimal way of the present invention, described method comprises: with described potential mass treatment cell, described cell expressing amyloid precursor protein or fragment, described albumen or fragment contain amyloid precursor protein exon 2 zone; Whether form patch structure in this cell of determination step, if form patch structure, then described potential material is transportation or the metabolic material that suppresses amyloid precursor protein.

The present invention has also comprised the transportation or the metabolic material that can be used for suppressing amyloid precursor protein that adopts described screening method to obtain, and described material is effective for alleviating nervus retrogression relative disease such as senile dementia.The present invention has also comprised the composition that contains described material, and the preparation of composition is easy to realize for those skilled in the art.

Major advantage of the present invention is:

(1) located a zone that plays an important role for the metabolism of amyloid precursor protein first, thereby provide valid approach for the medicine of toxicity molecule for screening suppresses the amyloid precursor protein metabolism.

(2) provide a kind of transportation of inhibition amyloid precursor protein or the effective ways and inhibitory substance of metabolic material of screening.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.

The acquisition of the APP of the disappearance of embodiment 1.E2

1.cDNA library screening and Southern are hybridized the preparation of used probe template

(1) preparation of cDNA library screening probe template (Probe 1)

(a) use TRIZOL reagent, from SK-N-SH cell (available from the Shanghai Inst. of Life Science, CAS cell bank), extract total RNA.

(b) ordinary method is carried out reverse transcription RT-PCR.

(c) be template with the RT-PCR product, use primer that P1/P2 (table 1) is carried out pcr amplification.

(d) electrophoresis reclaims the purifying pcr amplification product, and the clone advances the pGEM-T-easy carrier, and enzyme is cut, order-checking.

(e) Xho I and Sac I enzyme are cut, and the 682bp fragment that electrophoresis recovery purifying obtains promptly is Probe 1.

(2) Southern is hybridized the preparation of used probe template (Probe 2 and Probe 3)

(a) preparation of Probe 2: the APP695cDNA that obtains with the sieve storehouse is a template, uses primer P3/P4 (table 1) to carry out pcr amplification, and resulting 459bp fragment is Probe 2.

(b) preparation of Probe 3: cut the plasmid that contains APP695cDNA with Kpn I and Acc I enzyme, electrophoresis, the 167bp fragment that the recovery purifying obtains is Probe 3.

2.APP639cDNA acquisition

The inventor through two-wheeled hybridization, obtains 17 positive colonies with Probe 1 screening people tire brain cDNA library (available from GIBCO company) altogether.Utilizing primer P1/P6 (table 1) to carry out pcr analysis and enzyme cuts and identifies these clones.In 17 positive colonies, there are 14 clones to contain the encoding sequence of app gene, one of them is cloned corresponding to the APP770 type, and a clone is corresponding to the APP751 type, and all the other 12 clones belong to the APP695 type.The contriver has selected several clones corresponding to APP695 and has checked order, and found that Clone I3 except not containing the 7th, 8 exons, also lacks the 2nd exon, causes the 1st directly to be connected with the 3rd exon.Complete sequence determination shows that Clone I3 total length is 3011bp, reads frame and contains 1920bp, infers that encoded protein contains 639 amino acid (1-20 position among the SEQ ID NO:1,77-695 position).Therefore, contriver general's called after APP639.

Table 1

The disappearance of embodiment 2.E2 has caused the metabolic downward modulation of APP

The inventor has made up C and has held the expression plasmid of the different shear-forms of APP that have myc-tag, and construction process is as follows:

With APP695cDNA (referring to the GenBank accession number: NM_201414.1) be template, use 5 ' primer (5 '-TTT TCT AGAGAT GCT GCC CGG TTT G-3 ' (SEQ ID NO:8)) and 3 ' primer (5 '-GAG TCT AGACTA GTT CTG CAT CTG C-3 ' (SEQ ID NO:9)) (underscore is the site of Xba I) carries out pcr amplification.The purifying pcr amplified fragment uses Xba I (available from Japanese TAKARA company) enzyme to cut; Also use restriction enzyme Xba I enzyme to cut to carrier myc-pcDNA3 (purchasing company) simultaneously in American I nvitrogen, to be inserted into the myc-pcDNA3 after enzyme is cut through the described PCR fragment that enzyme is cut, obtain to contain the plasmid (APP695-myc carrier) of total length APP695 (being abbreviated as 695 among the figure) and myc-tag.

With APP639cDNA is template, uses 5 ' primer (5 '-TTT TCT AGAGAT GCT GCCCGG TTT G-3 ' (SEQ ID NO:10)) and 3 ' primer (5 '-GAG TCT AGACTA GTT CTGCAT CTG C-3 ' (SEQ ID NO:11)) (underscore is the site of Xba I) carries out pcr amplification.The purifying pcr amplified fragment uses Xba I enzyme to cut; Also use Xba I enzyme to cut to carrier myc-pcDNA3 (purchasing company) simultaneously in American I nvitrogen, to be inserted into the myc-pcDNA3 after enzyme is cut through the described PCR that enzyme is cut, acquisition contains the plasmid (APP639-myc carrier) of APP639 (promptly lack the APP695 of exon 2, be abbreviated as Δ E2 among the figure) and myc-tag.

Contain the APP695 (being abbreviated as Δ E3 among the figure) that lacks the 3rd exon and the plasmid construction of myc-tag: with APP695-myc is template, use 5 ' primer (5 '-TTG GTG AGT TTG TAA GTGATG CCC TTC TCG-3 ' (SEQ ID NO:12)) and 3 ' primer (5 '-TTC TTG GCA ATACTG CAG GAT GCC TTC CTT G-3 (SEQ ID NO:13)) to carry out pcr amplification, the purifying pcr amplified fragment, use Dpn I (available from U.S. NEB company) to digest unnecessary template, contain APP654 and (promptly lack the APP695 of E3 from getting continuously, APP-Δ E3 is abbreviated as Δ E3) and the plasmid (APP654-myc carrier) of myc-tag.

The recombinant plasmid transfection of above-mentioned structure is arrived in the 293T cell (available from the Shanghai Inst. of Life Science, CAS cell bank), by the detection of APP meta-bolites amount being investigated the metabolism situation of APP, in cell, to change empty carrier in contrast.Utilize Western Blot to detect the proteic expression of APP639.Utilize myc antibody (purchasing company) in American I nvitrogen, successfully detect the APP639-myc fusion rotein (among Fig. 2 A 2,5 swimming lanes), the APP654-myc fusion rotein is (among Fig. 2 A 3,6 swimming lanes) and among APP695-myc fusion rotein Fig. 2 A 4,7 swimming lanes), do not detect respective strap (1 swimming lane among Fig. 2 A) at control group.And the result shows the C99myc that the APP639-myc metabolism produces, and C83myc and AICDmyc fusion rotein will obviously be less than APP654-myc and APP695-myc.Also promptly, the APP695 of total length can produce more C end fragment (CTF); After having lacked E2, the level of CTF all obviously descends, and the amount of the A β in the corresponding cell training liquid also will be starkly lower than (Fig. 2 B) that APP695 produces, but lacks the metabolism not influence of the 3rd exon corresponding amino acid sequence to APP.

The The above results explanation, the disappearance of E2 can cause the metabolic downward modulation of APP specifically.

The disappearance of embodiment 3.E2 makes that the alpha-secretase enzyme of APP and beta-secretase pathways metabolism are all reduced

For the further molecular mechanism of this species specific downward modulation of research, the inventor at length investigated the α of APP-, the beta-secretase pathways metabolism.In most cases, APP is main in cell to be decomposed through alpha-secretase enzymes metabolism approach, after crossing expression beta-secretase (BACE), mainly carries out the beta-secretase metabolic process.The inventor has investigated the generation situation of the meta-bolites of APP respectively under the situation of not transfection and transfection BACE.

Method as embodiment 2, structure contains myc and APP total length, disappearance exon 2, lacks the plasmid of exon 3, transient transfection is gone into the HEK293 cell respectively, collect the training liquid of above-mentioned transfectional cell, precipitation albumen wherein utilizes the means of Western Blot to detect the proteic production of sAPP.

Discovery α-, in β-two pathways metabolism, CTF and sAPP that APP-Δ E2 metabolism is produced will be less than APP695.APP has a kind of Swedish (sw) mutant form, be that the KM before the shearing site of BACE is sported NL, this mutant form is easy to be sheared by BACE, often be used as the metabolic model of research APP (Forman MS etc., Differential effects of the swedish mutant amyloidprecursor protein on beta-amyloid accumulation and secretion in neurons andnonneuronal cells.J Biol Chem 1997; 272 (51): 32247-32253).The inventor has made up the APPsw-myc mutant form of disappearance E2 again, has obtained (Fig. 3 A, alpha-secretase enzymes metabolism: swimming

lane

2,3 with the similar result of APP695; Beta-secretase metabolism: swimming lane 8,9).

Utilize wo-2 antibody (available from the 6th medical college of Peking University) to detect the sAPP that the APP695-myc metabolism produces.Do not detect the sAPP that the APP639-myc metabolism produces, illustrate that the ability of APP639 metabolism generation sAPP will be weaker than APP695, see Fig. 3 A.

In order to observe APP and Secretases in intracellular location situation, the inventor has made up the APP-EYFP expression vector that has EYFP-tag respectively and has had the BACE-ECFP expression vector of ECFP-tag, and construction process is as follows:

APP639-EYFP: the EYFP-tag fragment is downcut from pEYFP (available from U.S. Clontech company) carrier with Xba I, and purifying enzyme is cut the back fragment, is inserted in the APP639-myc carrier after cutting with Xba I enzyme, obtains the APP639-EYFP expression vector.

APP695-EYFP: the EYFP-tag fragment is downcut from the pEYFP carrier with Xba I, and purifying enzyme is cut the back fragment, is inserted in the APP695-myc carrier after cutting with Xba I enzyme, obtains the APP695-EYFP expression vector.

BACE-ECFP: with BACE1cDNA (referring to the GenBank accession number: NM_012104) be template, use 5 ' primer (5 '-TCC TCT AGA ACT AGT GGA TCC ATG GCC CAA-3 ' (SEQ ID NO:14)) and 3 ' primer (5 '-GAG CTC GAG CTT CAG CAG GGA GATGTC ATC-3 ' (SEQ ID NO:15)) to carry out pcr amplification, obtain pcr amplified fragment, use XbaI and Xho I to carry out enzyme and cut, and purifying enzyme is cut the back fragment; Use Nhe I and Xho I enzyme to cut purification process to pECFP-N1 (available from U.S. Clontech company) simultaneously, aforementioned PCR fragment is inserted obtained the BACE-ECFP expression vector.

The recombinant vectors transfection of above-mentioned structure is arrived in the COS7 cell (available from the Shanghai Inst. of Life Science, CAS cell bank), observe APP639-EYFP respectively by Laser Scanning Confocal Microscope, APP695-EYFP and BACE-ECFP are in the intracellular situation of locating altogether, and white shows that two kinds of albumen have common location.The result shows that APP695-EYFP and BACE-ECFP can locate altogether in cell, APP639-EYFP (APP-Δ E-EYFP) and BACE-ECFP can not locate in cell altogether, see Fig. 3 B.

The disappearance of embodiment 4.E2 makes the Subcellular Localization of APP be affected

The inventor finds in the superincumbent experiment, and the amount of sAPP all obviously reduces in the APP meta-bolites, and therefore this metabolic downward modulation should be to occur in APP to be sheared this step by alpha-secretase enzyme or beta-secretase.But can't determine since APP and Secretases can not in conjunction with, still can not navigate on the corresponding organoid simultaneously due to.Therefore, the inventor has investigated the Subcellular Localization situation of the APP of different shear-forms again.

APP comprises secretion and two processes of endocytosis in intracellular transportation.In secretion process, APP can form sophisticated albumen through amination and glycosylated modification on endoplasmic reticulum and golgi body, so under the normal circumstances, APP has prematurity in cell, half ripe and sophisticated three kinds of forms.

As method construction recombination plasmid and the transfectional cell of embodiment 2, utilize Western Blot method to detect the proteic mature condition of APP639 in the interior golgi body of cell.Utilize myc antibody and wo-2 antibody, detect the mature form and the prematurity form of APP654-myc and APP695-myc fusion rotein.The band (Fig. 4 A, swimming lane 3,4) that these three kinds of form correspondences are arranged in the cell of can see transfection APP-Δ E3 and APP695 really, and transfection have only the band (Fig. 4 A, swimming lane 2) of immature APP correspondence in APP-Δ E2 and the cell.This explanation APP-Δ E2 does not navigate on the golgi body, and APP639myc can not normal mature, rests in the endoplasmic reticulum.

APP639-EYFP and APP695-EYFP are distinguished transfection in the COS7 cell, utilize the immunofluorescence dyeing method, use LMAN 1 (Univ Michigan-Ann Arbor USA doctor Zhang Bin provide), GM-130 (available from U.S. BD Bioscience company), EEA-1 (available from U.S. BD Bioscience company), M6PR (available from U.S. Santa Cruz company) antibody detects APP639-EYFP and APP695-EYFP and endoplasmic reticulum respectively to golgi body, golgi body, early stage endocytosis body, late period the endocytosis body common location situation.The result shows, APP-Δ E2-EYFP and endoplasmic reticulum to the transport vesicle of golgi body (Fig. 4 B, LMAN1) and golgi body (Fig. 4 B GM130) locatees altogether, and APP695-EYFP can well locate altogether (Fig. 4 BLMAN1, GM130).

In the endocytosis process, APP is taken back in the cell by the endocytosis body on cytolemma, so APP695-EYFP and endocytosis body (Fig. 4 B, EEA-1, M6PR) all have well location altogether, and APP639-EYFP and endocytosis body location (Fig. 4 B, EEA-1, M6PR) altogether.

The metabolism of embodiment 5.APP albumen n end structural damage causing APP and location are unusual

When the situation of research APP location, the inventor finds that APP-Δ E2-EYFP can form the accumulative patch structure in cell, and the albumen of some false foldings tends to form such structure.The inventor has analyzed the feature of E2 sequence, find wherein to have three Cys residues (aa38, aa62, aa73), can form disulfide linkage, and disulfide linkage has vital role for protein structure stable, the inventor adopts conventional method that these Cys residues are sported Gly respectively, destroy these disulfide linkage, investigated metabolism and the location situation of the APP of these sudden changes.

Use the method for Western Blot and immunofluorescence dyeing respectively, investigate the APP695-myc behind the above-mentioned rite-directed mutagenesis and the metabolism and the location situation of APP695-EYFP fusion rotein.Western Blot result shows that these destructurized APP695 are consistent with APP639 metabolism situation, and the CTF of generation is less than APP695 (wt) (Fig. 5 A, the alpha-secretase enzymes metabolism: swimming lane 4-6, beta-secretase metabolism: swimming lane 10-12) of not sudden change.

Immunostaining is the result also show, the APP695-myc of three halfcystine point mutation is the same with the APP639 fusion rotein with the APP695-EYFP fusion rotein in the APP695 albumen exon 2, can not eubolism, in cell, assemble agglomeratingly, form accumulative patch structure (Fig. 5 B).

The special construction that embodiment 6.APP-Δ E2 forms is not an aggregate

When producing a large amount of misfolded proteins in the cell, general degradation mechanism these albumen of can not degrading fully, will form the structure of aggregate (Aggresome), in this case, the albumen of false folding is taken to around the microtubule set center by scleroproein, formation is rich in the bag of ubiquitin by fibrinous structure, and this structure can not be dissolved in gentle washing agent.Therefore, can whether common location be arranged, and whether be dissolved in gentle washing agent and determine whether this special structure is aggregate by investigating APP-Δ E2 with ubiquitin and scleroproein.

With reference to the inferior localization method of the cell of embodiment 4, utilize the method for immunofluorescence dyeing, (γ-tubulin) and ubiquitin (Ubiquitin) antibody (all available from U.S. Santa Cruz company) are as the marker protein of aggregate, and whether investigate APP639-EYFP and APP695-EYFP albumen has common location with them to use anti-vimentin (Vimentin), γ-tubulin respectively.The result shows that APP639-EYFP and APP695-EYFP and these marker proteins are not all located altogether, and APP639 does not form aggregate, as Fig. 6 A.

With reference to the methods involving of embodiment 2, utilize the means of Western Blot to detect the distribution situation of APP639 albumen in washing agent (NP40) indissolvable component (i).Utilize anti-myc antibody and anti-wo-2 antibody, detect APP639-myc and APP695-myc fusion rotein and all distribute morely in washing agent (NP40) soluble component (s), it is less to distribute in the washing agent insoluble component, sees Fig. 6 B.And the APP639-myc fusion rotein only detects the prematurity form.The result shows that APP639 does not form aggregate.

The drug screening of embodiment 7. micromolecular compounds

Adopt X ray crystalline diffraction technology, protein three-dimensional structure according to the proteic extracellular region of APP695 (the N end contains 28-123 position among the SEQ IDNO:1) aminoacid sequence simulation, adopting high-throughput virtual screening technology, is candidate substances with the compound in the SPECS compound database.Have the compound of specificity keying action to sort out preliminary screening and APP exon 2 zone, the result has obtained some and has thought potential effective candidate compound through primary dcreening operation.

Embodiment 8. Antibody Preparation

Recombinant expressed and the purifying acquisition albumen of ordinary method, described protein sequence carries out animal immune with this albumen as antigen shown in 21-76 position among the SEQ ID NO:1.

Immune animal is New Zealand white rabbit, and is male, and body weight is 2-2.5kg.The immunity flow process: antigen is mixed with Freund ' s adjuvant at 1: 1, and after the emulsification, subcutaneous and intracutaneous multi-point injection carries out immunity.Antigenic dosage is 0.9mg/ time/rabbit.Complete Freund ' the s adjuvant of first immunisation, immunity full Freund ' the s adjuvant that toos many or too much for use once more.Be 3 weeks the pitch time of immunity.In 1 week of back of immunity for the third time, antiserum(antisera) is collected in the rabbit bloodletting, and how anti-the immunity that obtains anti-APP albumen E2 zone is.Utilize APP albumen as antigen, carry out the antigen antibody reaction checking with aforementioned acquisition how anti-, found that described many anti-can specificity in conjunction with the proteic E2 of APP zone, and debond other zone on foreign protein and APP albumen.

The antibody of above-mentioned preparation is joined in the 293T cell as the expression APP695 of the method preparation of embodiment 2, by the detection of APP meta-bolites amount being investigated the metabolism situation of APP, and with compare without the cell of the expression APP695 of antibody treatment, the cell that changes the cell of empty carrier over to and change the APP639 expression vector over to.Found that because the specificity sealing process of antibody, the metabolism situation of the cell APP after antibody treatment significantly is lower than the situation without antibody treatment, but suitable with the cell that changes the APP639 expression vector over to.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

＜120〉effect of amyloid precursor protein N-end structure in its transportation and metabolic process

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1 5 10 15

Ala?Leu?Glu?Val?Pro?Thr?Asp?Gly?Asn?Ala?Gly?Leu?Leu?Ala?Glu?Pro

20 25 30

Gln?Ile?Ala?Met?Phe?Cys?Gly?Arg?Leu?Asn?Met?His?Met?Asn?Val?Gln

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Asn?Gly?Lys?Trp?Asp?Ser?Asp?Pro?Ser?Gly?Thr?Lys?Thr?Cys?Ile?Asp

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Thr?Lys?Glu?Gly?Ile?Leu?Gln?Tyr?Cys?Gln?Glu?Val?Tyr?Pro?Glu?Leu

65 70 75 80

Gln?Ile?Thr?Asn?Val?Val?Glu?Ala?Asn?Gln?Pro?Val?Thr?Ile?Gln?Asn

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Trp?Cys?Lys?Arg?Gly?Arg?Lys?Gln?Cys?Lys?Thr?His?Pro?His?Phe?Val

100 105 110

Ile?Pro?Tyr?Arg?Cys?Leu?Val?Gly?Glu?Phe?Val?Ser?Asp?Ala?Leu?Leu

115 120 125

Val?Pro?Asp?Lys?Cys?Lys?Phe?Leu?His?Gln?Glu?Arg?Met?Asp?Val?Cys

130 135 140

Glu?Thr?His?Leu?His?Trp?His?Thr?Mal?Ala?Lys?Glu?Thr?Cys?Ser?Glu

145 150 155 160

Lys?Ser?Thr?Asn?Leu?His?Asp?Tyr?Gly?Met?Leu?Leu?Pro?Cys?Gly?Ile

165 170 175

Asp?Lys?Phe?Arg?Gly?Val?Glu?Phe?Val?Cys?Cys?Pro?Leu?Ala?Glu?Glu

180 185 190

Ser?Asp?Asn?Val?Asp?Ser?Ala?Asp?Ala?Glu?Glu?Asp?Asp?Ser?Asp?Val

195 200 205

Trp?Trp?Gly?Gly?Ala?Asp?Thr?Asp?Tyr?Ala?Asp?Gly?Ser?Glu?Asp?Lys

210 215 220

Val?Val?Glu?Val?Ala?Glu?Glu?Glu?Glu?Val?Ala?Glu?Val?Glu?Glu?Glu

225 230 235 240

Glu?Ala?Asp?Asp?Asp?Glu?Asp?Asp?Glu?Asp?Gly?Asp?Glu?Val?Glu?Glu

245 250 255

Glu?Ala?Glu?Glu?Pro?Tyr?Glu?Glu?Ala?Thr?Glu?Arg?Thr?Thr?Ser?Ile

260 265 270

Ala?Thr?Thr?Thr?Thr?Thr?Thr?Thr?Glu?Ser?Val?Glu?Glu?Val?Val?Arg

275 280 285

Val?Pro?Thr?Thr?Ala?Ala?Ser?Thr?Pro?Asp?Ala?Val?Asp?Lys?Tyr?Leu

290 295 300

Glu?Thr?Pro?Gly?Asp?Glu?Asn?Glu?His?Ala?His?Phe?Gln?Lys?Ala?Lys

305 310 315 320

Glu?Arg?Leu?Glu?Ala?Lys?His?Arg?Glu?Arg?Met?Ser?Gln?Val?Met?Arg

325 330 335

Glu?Trp?Glu?Glu?Ala?Glu?Arg?Gln?Ala?Lys?Asn?Leu?Pro?Lys?Ala?Asp

340 345 350

Lys?Lys?Ala?Val?Ile?Gln?His?Phe?Gln?Glu?Lys?Val?Glu?Ser?Leu?Glu

355 360 365

Gln?Glu?Ala?Ala?Asn?Glu?Arg?Gln?Gln?Leu?Val?Glu?Thr?His?Met?Ala

370 375 380

Arg?Val?Glu?Ala?Met?Leu?Asn?Asp?Arg?Arg?Arg?Leu?Ala?Leu?Glu?Asn

385 390 395 400

Tyr?Ile?Thr?Ala?Leu?Gln?Ala?Val?Pro?Pro?Arg?Pro?Arg?His?Val?Phe

405 410 415

Asn?Met?Leu?Lys?Lys?Tyr?Val?Arg?Ala?Glu?Gln?Lys?Asp?Arg?Gln?His

420 425 430

Thr?Leu?Lys?His?Phe?Glu?His?Val?Arg?Met?Val?Asp?Pro?Lys?Lys?Ala

435 440 445

Ala?Gln?Ile?Arg?Ser?Gln?Val?Met?Thr?His?Leu?Arg?Val?Ile?Tyr?Glu

450 455 460

Arg?Met?Asn?Gln?Ser?Leu?Ser?Leu?Leu?Tyr?Asn?Val?Pro?Ala?Val?Ala

465 470 475 480

Glu?Glu?Ile?Gln?Asp?Glu?Val?Asp?Glu?Leu?Leu?Gln?Lys?Glu?Gln?Asn

485 490 495

Tyr?Ser?Asp?Asp?Val?Leu?Ala?Asn?Met?Ile?Ser?Glu?Pro?Arg?Ile?Ser

500 505 510

Tyr?Gly?Asn?Asp?Ala?Leu?Met?Pro?Ser?Leu?Thr?Glu?Thr?Lys?Thr?Thr

515 520 525

Val?Glu?Leu?Leu?Pro?Val?Asn?Gly?Glu?Phe?Ser?Leu?Asp?Asp?Leu?Gln

530 535 540

Pro?Trp?His?Ser?Phe?Gly?Ala?Asp?Ser?Val?Pro?Ala?Asn?Thr?Glu?Asn

545 550 555 560

Glu?Val?Glu?Pro?Val?Asp?Ala?Arg?Pro?Ala?Ala?Asp?Arg?Gly?Leu?Thr

565 570 575

Thr?Arg?Pro?Gly?Ser?Gly?Leu?Thr?Asn?Ile?Lys?Thr?Glu?Glu?Ile?Ser

580 585 590

Glu?Val?Lys?Met?Asp?Ala?Glu?Phe?Arg?His?Asp?Ser?Gly?Tyr?Glu?Val

595 600 605

His?His?Gln?Lys?Leu?Val?Phe?Phe?Ala?Glu?Asp?Val?Gly?Ser?Asn?Lys

610 615 620

Gly?Ala?Ile?Ile?Gly?Leu?Met?Val?Gly?Gly?Val?Val?Ile?Ala?Thr?Val

625 630 635 640

Ile?Val?Ile?Thr?Leu?Val?Met?Leu?Lys?Lys?Lys?G1n?Tyr?Thr?Ser?Ile

645 650 655

His?His?Gly?Val?Val?Glu?Val?Asp?Ala?Ala?Val?Thr?Pro?Glu?Glu?Arg

660 665 670

His?Leu?Ser?Lys?Met?Gln?Gln?Asn?Gly?Tyr?Glu?Asn?Pro?Thr?Tyr?Lys

675 680 685

Phe?Phe?Glu?Gln?Met?Gln?Asn

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Claims

1. isolating amyloid precursor protein fragment, the aminoacid sequence of described protein fragments is shown in 21-76 position among the SEQID NO:1.

2. the purposes of the described protein fragments of claim 1 is used to prepare or screens the material of specific effect in this protein fragments.

3. purposes as claimed in claim 2 is characterized in that, described material is selected from: peptide, pdef polypeptide, plan peptide, non-peptide compound, carbohydrate, fat, antibody or antibody fragment, part, organic molecule, inorganic molecules and nucleotide sequence.

4. one kind is screened the transportation of inhibition amyloid precursor protein or the method for metabolic potential material, and described method comprises:

With the amyloid precursor protein that contains the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1 or fragment target spot as screening, select the material of specific effect in this target spot, described material is transportation or the metabolic potential material that suppresses amyloid precursor protein.

5. method as claimed in claim 4 is characterized in that, described method comprises:

6. method as claimed in claim 5 is characterized in that, described method comprises:

7. as the arbitrary described method of claim 4-6, it is characterized in that, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, further to select from candidate substances and to determine for the transportation or the useful material of metabolism that suppress amyloid precursor protein.

8. method as claimed in claim 7 is characterized in that, described method also comprises:

(4) the potential mass treatment cell that obtains with step (3), described cell expressing amyloid precursor protein or fragment, described albumen or fragment contain the aminoacid sequence shown in the 21-76 position among the SEQ ID NO:1;

(5) observe the interior amyloid precursor protein of cell of step (4) or the common location situation of fragment and endoplasmic reticulum, golgi body, transport vesicle or endocytosis body, if not having common location, then described potential material basically is transportation or the metabolic material that suppresses amyloid precursor protein.

9. method as claimed in claim 7 is characterized in that, described method also comprises:

The amount of amyloid precursor protein C end fragment in the cell of (5 ') determination step (4 '), and with control group relatively; Wherein said control group is expression amyloid precursor protein or the segmental cell without the potential mass treatment of step (3) acquisition;

If the amount of amyloid precursor protein C end fragment is lower than the amount of amyloid precursor protein C end fragment in the cellular control unit statistically in the cell of described potential mass treatment, then described potential material is transportation or the metabolic material that suppresses amyloid precursor protein.

10. method as claimed in claim 7 is characterized in that, described method also comprises:

11. transportation or metabolic material by the inhibition amyloid precursor protein of the arbitrary described method acquisition of claim 4-10.

12. the antibody of an anti-amyloid precursor protein, described antibodies specific ground is in conjunction with the described protein fragments of claim 1.