WO2009030094A1 - Fonction et utilisation d'une nouvelle protéine induisant une apoptose associée aux lysosomes contenant les domaines ph et fyve (lapf) - Google Patents

Fonction et utilisation d'une nouvelle protéine induisant une apoptose associée aux lysosomes contenant les domaines ph et fyve (lapf) Download PDF

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WO2009030094A1
WO2009030094A1 PCT/CN2007/070640 CN2007070640W WO2009030094A1 WO 2009030094 A1 WO2009030094 A1 WO 2009030094A1 CN 2007070640 W CN2007070640 W CN 2007070640W WO 2009030094 A1 WO2009030094 A1 WO 2009030094A1
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lapf
apoptosis
inducing protein
sensitivity
cells
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PCT/CN2007/070640
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Chinese (zh)
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Wei Chen
Nan Li
Xuetao Cao
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Zhejiang University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • LAPF novel human lysosomal-associated apoptosis-inducing protein
  • the present invention is in the field of biotechnology and medicine, and in particular, the present invention relates to a human lysosomal-associated apoptosis-inducing protein LAPF and uses thereof. Background technique
  • Apoptosis is a spontaneous death process that occurs after the body's cells undergo physiological and pathological stimulation. It has a very strong process in embryonic development, maintenance of mature organs, regulation of immune function, and tumorigenesis. Significance.
  • the nuclear fragmentation is mainly characterized by chromatin gradually collecting in the inner layer of the nuclear membrane to form a large chromatin mass with high electron density.
  • the nuclear membrane remains intact, followed by fragmentation at multiple locations, the nucleus becoming smaller, and finally cleavage into several densely stained fragments. Nuclear fragmentation eventually leads to cell death.
  • the object of the present invention is to provide a novel apoptosis-inducing protein LAPF protein and its function.
  • an apoptosis-inducing protein LAPF or a gene encoding the same or an agonist thereof for the preparation of a composition for increasing the sensitivity of a cell to tumor necrosis factor alpha (; TNFa).
  • the composition is for promoting nuclear fragmentation of cells.
  • the composition is for promoting apoptosis.
  • the apoptosis inducing protein LAPF is selected from the group consisting of:
  • a protein derived from (1) which is formed by substitution, deletion or addition of the amino acid sequence of SEQ ID NO: 2 via one or more amino acid residues, and which has a function of regulating apoptosis.
  • composition comprising:
  • the composition is for promoting apoptosis.
  • the composition is a pharmaceutical composition.
  • composition further comprises:
  • LAPF apoptosis-inducing protein
  • a method of screening for a potential substance that increases the sensitivity of a cell to tumor necrosis factor a comprising the steps of:
  • step (a) comprises: adding a candidate substance to the system containing the apoptosis-inducing protein LAPF in the test group; and/or
  • the step (b) comprises: detecting the expression or activity of the apoptosis-inducing protein LAPF in the system of the test group, and comparing the control group to the control group containing the apoptosis-inducing protein LAPF without adding the candidate substance. ;
  • the control group indicates This candidate is a potential substance that increases the sensitivity of cells to tumor necrosis factor alpha.
  • the method further includes the steps of:
  • system is a cellular system.
  • a method of increasing the sensitivity of a cell to tumor necrosis factor alpha in vitro (therapeutic or non-therapeutic) or in vivo characterized in that the method comprises: increasing the apoptosis-inducing protein LAPF in the cell Expression or activity.
  • Figure 1 shows the distribution shown by human LAPF Northern blot hybridization of the present invention. The results suggest that LAPF is a widely distributed intracellular molecule.
  • Fig. 2 shows an RT-PCR analysis chart of the human LAPF of the present invention.
  • FIG. 3 shows that transient overexpression of human LAPF protein induces apoptosis in L929 cells.
  • the picture shows the Annexin V/PI staining results.
  • Figure 4 shows that stable overexpression of human LAPF protein increases the sensitivity of L929 cells to TNF d.
  • A is the Western result of LAPF overexpression;
  • B is the Annexin V/PI staining result;
  • C is the scanning electron micrograph.
  • the inventors discovered for the first time that the apoptosis-inducing protein LAPF can increase the sensitivity of cells to tumor necrosis factor a CTNF c. Moreover, the inventors have also found that the LAPF can also promote nuclear fragmentation or apoptosis of cells. Therefore, the apoptosis-inducing protein LAPF itself can be used as a substance for increasing the sensitivity of cells to TNF ci, and the apoptosis-inducing protein LAPF can be used as a target for screening to increase cells for TNF a by promoting its activity or expression. Sensitive substance. The present invention has been completed on the basis of this.
  • the present inventors isolated a novel molecule from normal human bone marrow stromal cells and found that it has a function of inducing apoptosis such as L-929, and can translocate from cytoplasm to lysosome during apoptosis. In vivo, it is named LAPF (Lysosome-assocated Apoptos i s- induc ing prote in containing PH and FYVE domains).
  • LAPF Lysosome-assocated Apoptos i s- induc ing prote in containing PH and FYVE domains.
  • LAPF contains a platelet-leukocyte C kinase substrate homologous domain (PH) and a FYVE zinc finger domain. These two domains do not have phosphorylation sites, but are closely related to protein localization and binding. Hydrophobic analysis of LAPF revealed no significant signal peptides and transmembrane regions, presumably a cytosolic protein. Therefore, LAPF may act as a linker protein in certain signal transduction pathways. By homologous analysis, it was found that 13 unknown proteins have high homology with LAPF, and they all contain only two domains, ⁇ 3 ⁇ 4 and FYVE, so the inventors classified these proteins into a new family. Phaf ins (prote in containing both PH and FYVE domains).
  • LAPF is predominantly expressed in the heart and skeletal muscle of normal adults, and liver, placenta, lung, and peripheral blood lymphocytes are also expressed at low levels.
  • RT-PCR analysis showed that LAPF is highly expressed in peripheral blood monocyte-derived dendritic cells, and is widely expressed in various tumor cells such as hematopoietic tumors, but not in solid tumor cell lines such as breast cancer.
  • LAPF can induce apoptosis of L929 (mouse fibroblasts) without the synergistic effect of LPS or TNF-ci.
  • stable and high expression of LAPF can also increase the sensitivity of L929 cells to TNF- ⁇ .
  • the inventors observed the translocation process of LAPF from cytosol to lysosome by confocal microscopy. Further studies have found that LAPF can induce lysosomal membrane instability during apoptosis, indicating that LAPF achieves apoptosis-promoting through the lysosomal pathway. Further studies have also found that LAPF can increase the sensitivity of cells to TNF alpha and promote cell apoptosis.
  • LAPF is a novel intracellular molecule that plays an important role in the signal transduction of apoptosis.
  • LAPF has important development and application value in many fields such as regulation of immune function and inhibition of tumor growth.
  • LAPF protein apoptosis-inducing protein LAPF
  • LAPF polypeptide apoptosis-inducing protein LAPF
  • apoptosis-inducing protein LAPF refers to a mammalian apoptosis-inducing protein LAPF amino acid sequence having a human-containing ⁇ 3 ⁇ 4 domain and a FYVE domain (SEQ ID NO : 2) A protein or polypeptide.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • isolated LAPF protein or polypeptide means that the LAPF polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Those skilled in the art will be able to purify the LAPF protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptides of the invention may be naturally purified products, either chemically synthesized or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).
  • the polypeptide of the invention may be glycosylated according to the host used in the recombinant production protocol, or may be non-sugar Basic. Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human LAPF proteins.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the native human LAPF protein of the present invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or (iv) an additional amino acid sequence fused to the polypeptide sequence (eg, a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic IgG fragment).
  • conservative amino acid residues preferably conservative amino acid residues
  • human LAPF polypeptide refers to a polypeptide having the sequence of SEQ ID NO: 2 of human LAPF protein activity.
  • the term also encompasses variant forms of the sequence of SEQ ID NO: 2 that have the same function as the human LAPF protein. These variants include, but are not limited to, a number (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions And/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is usually not altered.
  • the addition of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the human LAP F protein.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize to human LAPF DNA under high or low stringency conditions And a polypeptide or protein obtained using an antiserum against a human LAPF polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising a human LAPF polypeptide or a fragment thereof.
  • the present invention also encompasses soluble fragments of human LAPF polypeptides.
  • the fragment has at least about 10 contiguous amino acids of the human LAPF polypeptide sequence, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, optimally at least about 100 consecutive amino acids.
  • the invention also provides analogs of human LAPF proteins or polypeptides.
  • the difference between these analogs and the native human LAPF polypeptide may be a difference in amino acid sequence or a difference in the modification form that does not affect the sequence. Different, or both.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the "human LAPF protein conservative variant polypeptide” means up to 10, preferably up to 8, more preferably up to 5, most preferably up to the amino acid sequence of SEQ ID NO: 2.
  • the three amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be either a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1 or may be a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1.
  • Polynucleotides encoding the mature polypeptide of SEQ ID NO: 2 include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and Non-coding sequence.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include a polynucleotide encoding additional and/or non-coding sequences.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, Deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C ; or 2) Hybridization is carried out with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficol l , 42 ° C, etc.; or (3) only in two sequences Hybridization occurs when the identity is at least 90% or more, more preferably 95% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide represented by SEQ ID NO: 2.
  • Antagonists or agonists of LAPF and uses thereof
  • the invention also relates to antagonists and agonists of LAPF.
  • an antagonist of LAPF refers to a substance that is capable of combating, or reducing, the activity or expression of a LAPF polypeptide.
  • Common antagonists are antibodies, antisense nucleotides, and interfering RNA (RNAi). The antagonists can be used to reduce the sensitivity of cells to TNF[alpha].
  • an agonist of LAPF refers to any substance that increases the activity of LAPF, maintains the stability of LAPF, promotes LAPF expression, prolongs the effective duration of LAPF, or promotes transcription and translation of LAPF. These substances can be used to increase the sensitivity of cells to TNF a. Screening for substances that increase the sensitivity of cells to TNF a
  • the present invention provides a method of screening for potential substances that increase the sensitivity of a cell to TNFa, including the following steps:
  • the candidate substance is contacted with a system containing LAPF; the effect of the candidate substance on the expression or activity of LAPF is observed; wherein, if the candidate substance can increase the expression or activity of LAPF, it indicates that the candidate substance is to increase the sensitivity of the cell to TNF a Potential substance.
  • the system is selected from (but not limited to): solution system, cell system, sub-fine Cell system, tissue system, organ system, or animal system.
  • a control group in order to make it easier to observe a change in LAPF, a control group may be provided, and the control group may be a LAPF-containing system to which the candidate substance is not added.
  • These initially screened materials can constitute a screening library for further cell and/or animal testing of these materials so that they can ultimately be screened for drugs that are truly useful for increasing the sensitivity of cells to TNF[alpha].
  • the present invention also encompasses a class of substances obtained by the screening method of the present invention which increase the sensitivity of cells to TNF ⁇ , which act on LAPF, enhance LAPF activity, maintain LAPF stability, promote LAPF expression, and prolong LAPF. Effective action time, or promote the transcription and translation of LAPF to play a role in increasing the sensitivity of cells to TNF ci.
  • Compositions and methods of treatment which increase the sensitivity of cells to TNF ⁇ , which act on LAPF, enhance LAPF activity, maintain LAPF stability, promote LAPF expression, and prolong LAPF.
  • the present invention also provides a composition comprising an effective amount (e.g., 0.000001 to 0.1 g / 60 kg body weight / day; preferably, 0.00001 to 0.05 g / 60 kg body weight / day) of said LAPF protein, or
  • An agonist of LAPF and a pharmaceutically or food acceptable carrier.
  • the composition further comprises an effective amount of TNF (e.g., 0.000001 to 0.1 g / 60 kg body weight / day; preferably, 0.00001 - 0.05 g / 60 kg body weight / day).
  • TNF e.g., 0.000001 to 0.1 g / 60 kg body weight / day; preferably, 0.00001 - 0.05 g / 60 kg body weight / day.
  • LAPF protein increases the sensitivity of cells to TNF a after administration to cells.
  • a "pharmaceutically or foodly acceptable” ingredient is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, has reasonable benefits/ The substance of the risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceuti cal Scences (Mack Pub. Co., N. J. 1991).
  • the pharmaceutically acceptable carrier in the composition may contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the invention also provides a method of increasing the sensitivity of a cell to TNFa comprising administering to the subject an effective amount of a LAPF protein, or an agonist of LAPF.
  • a LAPF protein or an agonist of LAPF.
  • the effective dose of LAPF may vary depending on the severity of the subject to be treated. The specific circumstances are determined by the individual condition of the subject, and these factors are A skilled physician or dietitian can judge the scope.
  • LAPF and/or TNF ci can be directly administered to a subject by a method such as injection; or, an expression unit carrying LAPF and/or TNF a (such as an expression vector or a virus, etc.) can be delivered by a certain route.
  • an expression unit carrying LAPF and/or TNF a such as an expression vector or a virus, etc.
  • the specific circumstances depend on the type of agonist described, which are well known to those skilled in the art.
  • a gene encoding LAPF and/or TNFa, or a vector carrying the gene can be introduced into a target cell or a target tissue by a conventional method to effect expression of LAPF and/or TNF protein.
  • the target cells are tumor cells.
  • LAPF can significantly increase the sensitivity of cells to TNF ci.
  • LAPF itself can be used to prepare compositions that increase the sensitivity of cells to TNF ci, or LAPF can be used as a target for drug screening for screening for substances that increase the sensitivity of cells to TNF ci by promoting their activity or expression.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.
  • Example 1 Cloning of human LAPF cDNA
  • RNA from human bone marrow stromal cells was extracted with Trizol (Gibco). Then, poly(A) mRNA is isolated from total RNA. After poly(A) mRNA was reverse transcribed to form cDNA, the cDNA fragment was inserted into the multiple cloning site of the vector by SuperScriptll cloning kit (purchased from Gibco), and DH50C bacteria were transformed to form a cDNA plasmid library. The sequence at the 5' end of the randomly selected clone was determined by the dideoxy method. Comparing the determined cDNA sequence with an existing public DNA sequence database, and found a cDNA The cloned DNA sequence is a new full-length cDNA.
  • the DNA sequence contained in the new clone was bidirectionally determined by synthesizing a series of primers. Computer analysis indicated that the full-length cDNA contained in the clone was a new cDNA sequence (sequence shown in SEQ ID NO: 1) encoding a new protein (sequence as shown in SEQ ID NO: 2). This protein is called human LAPF and its coding gene is the human LAPF gene.
  • Sequence SEQ ID NO: 1 is 1698 bp in length and includes a 70 bp 5' non-coding region and a 788 bp 3' non-coding region encoding a polypeptide of 279 amino acids. It is theoretically calculated that the molecular weight of the unglycosylated mature molecule is about 30 kD.
  • LAPF differs from known genes in that it contains a ⁇ 3 ⁇ 4 domain and a FYVE zinc finger structure in the LAPF protein, which further suggests that it is a novel linker protein involved in cell signal transduction.
  • Example 2 Cloning the coding sequence of human LAPF by RT-PCR
  • RNA in the logarithmic growth phase B lymphoma cell line Raj i was extracted with Trizol (Gibco), and 6 mg of total RNA was mixed with 0.5 g of 01 igo-dT 12 - 18 for reverse transcription.
  • the reverse transcription system was 20 ⁇ l, and after the reaction was completed, 80 ⁇ l dd 0 was added for dilution.
  • the primers used for PCR amplification are as follows:
  • Sense primer 5 '-GCGAATTCATGGTGGACCACTTGGCCAACA-3 ' (SEQ ID NO: 3), antisense primer: 5, -GCAAGCTTTCAGCTGTGGAAAGCAGACCA-3 ' (SEQ ID NO: 4).
  • the PCR reaction volume was 50 ⁇ l, including the reverse transcription template 10 ⁇ l, 0.4 mM primer, 0.2 mM dNTP, and 1 U ExTaq DNA polymerase (Takara Inc.), and the amplification parameters were 94 ° C for 30 seconds and 60 ° C for 30 seconds. At 72 °C for 45 seconds, the PCR products were confirmed by 2% agarose gel electrophoresis after 25 cycles. The result of DNA sequence analysis revealed that the DNA sequence of the PCR product was identical to that of 1-1698 shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of LAPF
  • Northern blotting was performed as follows: The filter to be tested was placed in 10 ml of a pre-warmed hybridization solution at 68 ° C, pre-hybridized in a hybridization oven (Bel lco) at 68 ° C for 30 minutes; the labeled cDNA probe was Denature at 95 ⁇ 100 °C for 2 ⁇ 5 minutes, add rapidly to the ice and add the hybridization solution (final concentration of cDNA probe is 2 ⁇ 10ng/ml or l ⁇ 2 X 10 6 cpm/ml), mix well, at 68 Hybridize at °C for 2 hours.
  • the filter was rinsed several times with 2xSSC, 0.05% SDS at room temperature, followed by shaking for 30 to 40 minutes, during which the lotion was changed several times. Subsequently, it was washed with 0. lxSSC, 0.1% SDS at 50 ° C for 20 to 40 minutes. Finally, the filter was wrapped in a plastic wrap and exposed to X-ray film at -70 ° C for 24 to 48 hours.
  • the reverse transcription system was 20 ⁇ l, and after the reaction was completed, 80 ⁇ l dd 0 was added for dilution.
  • the primers used for PCR amplification of CaM-KIIN are as follows:
  • Sense primer 5 ' CGACGAGGACAAGGAGGGCAGCAGG (SEQ ID NO: 5),
  • Antisense primer 5 ' GTGGGTGTTCTGGGGCTTCCTGAAG (SEQ ID NO: 6) ,
  • ⁇ -actin was used as a positive control.
  • the PCR reaction volume was 50 ⁇ l, including the reverse transcription template 10 ⁇ 1, 0.5 mM primer, 0.2 mM dNTP, and 1 U rTaq DNA polymerase (Takara Inc.), and the amplification parameters were 95 ° C for 15 seconds, 57 ° C for 30 seconds. After 72 cycles of 72 °C, the PCR product was confirmed by 1. 5% agarose gel electrophoresis.
  • the DNA sequence of the PCR product was identical to that of 179-589 shown in SEQ ID NO: 1.
  • the human LAPF protein prepared by the conventional method is used to immunize an animal to produce an antibody, and the specific method is as follows.
  • the recombinant molecules are separated by chromatography and used. Separation can also be carried out by SDS-PAGE gel electrophoresis, and the electrophoresis band is excised from the gel and emulsified with an equal volume of complete Freund's adjuvant.
  • Mice were intraperitoneally injected with 50-100 ⁇ g/0.2 ml of the emulsified protein. After 14 days, the mice were intraperitoneally injected with a dose of 50-100 g/0.2 ml with the same antigen emulsified with a non-complete Freund's adjuvant to enhance the immunization.
  • Example 6 Construction of human LAPF eukaryotic expression vector and transfection of eukaryotic gene
  • amplification was carried out using PCR oligonucleotide primers at the 5' and 3' ends of the sequence below to obtain human LAPF DNA as an insert.
  • the 5'-end oligonucleotide primer sequence used in the PCR reaction is:
  • the obtained PCR product was purified and digested with EcoR I-Hind III and then ligated with eukaryotic expression vector plasmid pcDNA3.1/ myc-His (-) B (Invitrogen) was recombined and transformed into competent E. coli DH5a by conventional methods, and positive clones were picked for restriction enzyme digestion, purified and sequenced (ABI's Model 377 Sequencer, BigDye Terminator Kit, PE) the company). It was confirmed by sequencing that the complete LAPF coding sequence was inserted.
  • the LAPF eukaryotic expression plasmid DNA was transfected into human breast cancer MCF-7 cells, human lung cancer A549 cells and mouse fibroblast L929 (all obtained from ATCC) by liposome LipofectAMINE reagent (Invitrogen), with pcDNA3.1.
  • the plasmid vector served as an irrelevant control.
  • the main steps are as follows: The plasmid DNA to be transfected is mixed with liposome LipofectAMINE in a certain ratio and allowed to react at room temperature for 45 minutes; 60-80% confluent cells grown in 6-well cell culture plates, serum-free with 0PTI-MEM After washing the medium (Invitrogen) twice, add the plasmid DNA-liposome mixture, incubate at 37 °C 5% CO 2 for 6-8 hours, add an equal volume of normal medium containing 20% serum, and continue to culture for 6 hours. Replace the fresh medium afterwards. Transient expression was collected 48 hours after transfection and subjected to Western blot analysis to detect transfection effects.
  • Example 7 Western blot detection
  • L929 cells stably overexpressing LAPF protein were lysed with cell lysate (Cell Signaling) in Example 6.
  • the supernatant was taken at 13,000 rpm x lOmin by centrifugation at 4 ° C, and protein quantification was performed using a BCA protein assay kit (PIERCE).
  • the protein samples were subjected to SDS-PAGE, followed by transfer to a nitrocellulose membrane (Schleicher & Schuell) at a constant voltage of 100 V at 4 ° C, stained with Ponceau and labeled with size and orientation. Block at room temperature for 2 hours (5% skim milk powder in TBST solution), dilute the primary antibody with blocking solution, and incubate for 1 hour at room temperature.
  • TBST (0.05% Tween 20 in TBS solution) was washed for 15 minutes, 3 times, and the secondary antibody was diluted with blocking solution and incubated for 2 hours at room temperature.
  • the TBST was washed for 15 minutes, 3 times, and washed with TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 15 minutes, then added with a chemiluminescent substrate (Pierce) for 1 min, and rapidly sealed and auto-developed.
  • the primary antibody used for Western blot detection was the anti-LAPF antibody obtained in Example 5.
  • the secondary antibody was HRP-labeled anti-rabbit IgG (Cell Signaling).
  • LAPF expression product hLAPF
  • Example 8 Detection of LAPF-induced apoptosis activity
  • Apoptosis was detected using the Annexin V/FITC kit (Bender), and the procedure was as follows. Briefly described as follows: L929 cells overexpressing LAPF protein in Example 6 were washed with PBS and then resuspended in binding buffer. In the dark at room temperature, stain with Annexin V/ FITC for 10 minutes, wash with PBS and resuspend in binding buffer again, add PI, immediately load, analyze by flow cytometry, and analyze software is ModFi t 3 . 0 LTTM Software.
  • the morphological observation of apoptosis by transmission electron microscopy is as follows: The L929 cells overexpressing the LAPF protein in Example 6 were collected, and buffered with 0.1 M phosphate containing 2.5% glutaraldehyde. The solution was fixed at 4 ° C for 2 h in a solution (pH 7.4), and the cell pellet was washed twice with PBS, and fixed at 1 ° C for 1 h in 0.1 M phosphate buffer (pH 7.4) containing 1% citric acid.
  • Example 9 Screening for substances that increase the sensitivity of cells to TNF a
  • the L929 cells overexpressing LAPF described in Example 8 were used as test subjects, and the expression of LAPF in the cells before and after the stimulation of the candidate substance was detected, and the anti-human LAPF antibody prepared above was used for the hybridization test to detect the LAPF-LAPF antibody. The strength of each other is combined to know the expression changes of LAPF in the cells.
  • Test group L929 cells with candidate substance added, the cells overexpressing LAPF;
  • Control group L929 cells without candidate substance added, which overexpresses LAPF.

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Abstract

La présente invention concerne une composition qui renferme une protéine induisant une apoptose LAPF (protéine induisant une apoptose associée aux lysosomes contenant les domaines PH et FYVE). La composition peut être utilisée pour amplifier l'apoptose cellulaire.
PCT/CN2007/070640 2007-09-06 2007-09-06 Fonction et utilisation d'une nouvelle protéine induisant une apoptose associée aux lysosomes contenant les domaines ph et fyve (lapf) WO2009030094A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2007/070640 WO2009030094A1 (fr) 2007-09-06 2007-09-06 Fonction et utilisation d'une nouvelle protéine induisant une apoptose associée aux lysosomes contenant les domaines ph et fyve (lapf)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2007/070640 WO2009030094A1 (fr) 2007-09-06 2007-09-06 Fonction et utilisation d'une nouvelle protéine induisant une apoptose associée aux lysosomes contenant les domaines ph et fyve (lapf)

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WO2009030094A1 true WO2009030094A1 (fr) 2009-03-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114231551A (zh) * 2021-12-24 2022-03-25 云南大学 蛋白在促进昆虫淋巴细胞凋亡和/或防治害虫中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1093A (ja) * 1996-04-19 1998-01-06 Japan Found Cancer Res アポトーシス誘導タンパク質およびそれをコードする遺伝子
CN1475502A (zh) * 2002-08-16 2004-02-18 浙江大学免疫学研究所 新的凋亡诱导蛋白，其编码序列及用途
US20040224389A1 (en) * 1994-05-27 2004-11-11 The Regents Of The University Of Colorado Viral vectors encoding apoptosis-inducing proteins and methods for making and using the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040224389A1 (en) * 1994-05-27 2004-11-11 The Regents Of The University Of Colorado Viral vectors encoding apoptosis-inducing proteins and methods for making and using the same
JPH1093A (ja) * 1996-04-19 1998-01-06 Japan Found Cancer Res アポトーシス誘導タンパク質およびそれをコードする遺伝子
CN1475502A (zh) * 2002-08-16 2004-02-18 浙江大学免疫学研究所 新的凋亡诱导蛋白，其编码序列及用途

Non-Patent Citations (1)

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Title
PAOLA SECCHIERO ET AL.: "Systemic Tumor Necrosis Factor-Related Apoptosis-Inducin; Ligand Delivery Shows Antiatherosclerotic Activity in Apolipoprotein E-Null Diabetic Mice", CIRCULATION, vol. 114, no. 14, 2006, pages 1522 - 1530, XP055351586 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114231551A (zh) * 2021-12-24 2022-03-25 云南大学 蛋白在促进昆虫淋巴细胞凋亡和/或防治害虫中的应用
CN114231551B (zh) * 2021-12-24 2023-09-29 云南大学 蛋白在促进昆虫淋巴细胞凋亡和/或防治害虫中的应用

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