Top > Search of International Patents > PROBE REAGENT FOR MEASUREMENT OF PROTEIN DECOMPOSITION ACTIVITY

PROBE REAGENT FOR MEASUREMENT OF PROTEIN DECOMPOSITION ACTIVITY

Foreign code F110004996
File No. E08804WO
Posted date Aug 3, 2011
Country WIPO
International application number 2011JP051089
International publication number WO 2011090159
Date of international filing Jan 21, 2011
Date of international publication Jul 28, 2011
Priority data
  • P2010-012084 (Jan 22, 2010) JP
Title PROBE REAGENT FOR MEASUREMENT OF PROTEIN DECOMPOSITION ACTIVITY
Abstract A probe reagent comprising, from the N-terminal to the C-terminal, an amino acid sequence for a fluorescent protein I, an amino acid sequence for a peptide capable of terminating the decomposition of a protein (i.e., a decomposition termination peptide), an amino acid sequence for a spacer peptide, an amino acid sequence for a fluorescent protein II, and an amino acid sequence for a protein to be decomposed, wherein the probe reagent is characterized in that the protein to be decomposed is a protein capable of being decomposed with a ubiquitin-proteasome system and the probe reagent is decomposed from the C-terminal side but the decomposition is terminated at the decomposition termination peptide; a nucleic acid encoding the probe reagent; and use of the probe reagent or the nucleic acid.
Outline of related art and contending technology BACKGROUND ART
1 Cells as one of the protein with a pathway, ubiquitin - proteasome system is well known. In this reaction system, the denatured protein or abnormal protein folding and a plurality of small protein called ubiquitin, added to the chain. This ubiquitin chains as a marker of the protein should be decomposed and, degradation of the protein recognized by the proteasome device destruction. This system, the removal of the abnormal protein in a cell is performed. However, the ubiquitin - proteasome system, within the cell not only for the purpose of quality control of the system is not adopted. Structurally, functionally normal protein receptor protein, at that time and to degrade in response to the status of a cell, a decrease in the activity of the control is performed in the various cell functions. As a result of the large protein ubiquitin - proteasome system is controlled by the amount present is known. As the function of these proteins, cell cycle control, regulation of gene expression, stress response, in a wide variety restoration of DNA. In this way, a biological phenomenon in many cells is controlled by this system can be made, ubiquitin - proteasome system maintenance of the normal activities of the cells being essential. Therefore, deterioration of the function of this proteolytic system affect the critical to the cell. Alzheimer's disease is a neurodegenerative disease is Parkinson's disease, intracellular accumulation of abnormal protein aggregation is observed, as the cause of the onset of these diseases of the ubiquitin - proteasome system dysfunction has been suggested. The ubiquitin - proteasome system is, to be involved in the restoration of cell cycle or from DNA, canceration of cells from the failure of this system is induced also known. In this way, the ubiquitin - proteasome system a number of important biological phenomenon from the responsible for the control, the reaction was allowed to precisely and easily develop a method for measuring, life phenomena exhibited by elucidation of mechanism as well as, methods of treatment for disease induced by the abnormality in the development of chemical agents and the like greatly contribute to appear.
Ubiquitin - proteasome system performed in a cell of the measurement of the degradation of the protein, conventional, of the biochemical techniques such as Western blotting has been used. In this method a large number of cells are collectively destroyed, to recover components thereof. An object to be measured contained in the protein, depending on the molecular weight by electrophoresis separation, using antibody specific for further detected by staining. As a result of protein abundance and degradation activity is measured. However, this method is troublesome and time-consuming operation and then, the measurement of living in individual cells cannot be performed.
In recent years, and the like related to the firefly bioluminescent reaction luciferin, luciferase, fluorescent protein that is obtained from the aequorea victoria, for monitoring the kinetics of intracellular molecules of probe reagent as application techniques greatly developed. This is in combination with the advances in the techniques of microscopic imaging, a particular physiological activity within the cell to visualize the space-time, a method for measuring have been generalized. Activity of the proteasome for degradation of the protein, in a living cell by this method has been measured. In this method, a measuring object and luciferase or a protein fuzed to the fluorescent protein and the probe reagent (patent document 1). When the protein is present in cells, light emitting label or a fluorescence is observed, when proteasome degradation of the luciferase or fluorescent protein fuzed together and also can be decomposed into fellow traveler, not the luminescence or fluorescence was observed. Therefore, these variations in light intensity monitor of the cracking activity of this protein can be measured. So far by this method, IκB α, p27, p53, degradation of the protein such as HIF-1α measurement is being performed (patent document 2, non-patent document 1-3). However, these to be used as markers of probe reagent is luciferase or fluorescent protein is from the two types of 1, only the luminescence or fluorescence 1 measured by the wavelength. For this reason, the intensity of light, of the expression amount of non-uniform distribution of the probe reagent, in the form of cells and tissues, such as quenching by fading, such as unevenness of the excitation light does not depend on the degradation of the protein activity can be affected by factors such as, quantitative measurement is difficult. Increases the proteolytic activities that also decreases when the amount of the light intensity, of the short of the sensitivity of the detector resulting in a reduction in S/N, a precise measure is also a problem that it is difficult to.
1 The photometric method of wavelength only becomes a problem in degradation of the protein does not depend on the adverse effects of the factors is, the luminescence or fluorescence measured at a wavelength 2, that the mechanical strength of the second fuel can be cancelled by setting the ratio. In the method of Davis et al., protein as the object of measurement, the light emission of a click beetle green IκB α derived from fuzed to luciferase (CBG68) and expressed in a cell. At the same time click beetle-derived luciferase (CBR) red light emission of the expressed in the same cell. CBG68 Emits light in a quantity, according to the activity of the proteasome degradation IκB α showing the increase or decrease. On the other hand, the emission will be affected not CBR was used as control. They are cultured in multi-well plates at a cell population was measured and, due to a difference between the number of cells between wells in the amount of the light emitted in order to correct, red/green IκB α of the cracking activity measured as the ratio of the emission intensity of the (non-patent document 4). In this approach, the photometric method compared with the wavelength 1 is improved when it is quantitative, one 2 for the expression of probe molecules to individual, between cells of the expression amount of these ratio, that is the ratio of the difficult to have the same problem. Also be used as markers depending on the nature of the fluorescent protein luciferase, which are not uniformly distributed within the cells of the localized difference might be the performance of computer systems. These are, in particular at the single cell level of degradation of the protein activity was measured and if it is desired to become factors that reduce the accuracy.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 N末側からC末端側に向かって順番に、蛍光蛋白質I、蛋白質の分解を停止させるペプチド(分解停止ペプチド)、スペーサーペプチド、蛍光蛋白質II、および分解を受ける蛋白質のそれぞれのアミノ酸配列を含んでなるプローブ試薬であって、ここで該分解を受ける蛋白質がユビキチン-プロテアソーム系で分解される蛋白質であり、および、該プローブ試薬のC末端側から分解を受けるがその分解は分解停止ペプチドで停止することを特徴とするプローブ試薬。

[請求項2]
 蛍光蛋白質Iおよび蛍光蛋白質IIは、励起波長、蛍光波長、または、励起波長と蛍光波長の両方が異なるものである、請求項1に記載のプローブ試薬。

[請求項3]
 蛍光蛋白質Iおよび蛍光蛋白質IIはそれぞれ、蛍光エネルギー移動(FRET)でのドナーとアクセプタである、請求項1または2に記載のプローブ試薬。

[請求項4]
 核局在化シグナルまたは核外搬出シグナルをさらに含む、請求項1~3のいずれか1項に記載のプローブ試薬。

[請求項5]
 分解停止ぺプチドが、蛍光蛋白質Iとスペーサーの間に1個もしくは複数個存在する、請求項1~4のいずれか1項に記載のプローブ試薬。

[請求項6]
 スペーサーペプチドは、分解停止ペプチドと蛍光蛋白質IIの間に距離をおくための1個以上のアミノ酸からなるペプチドである、請求項1~5のいずれか1項に記載のプローブ試薬。

[請求項7]
 請求項1~6のいずれか1項に記載のプローブ試薬をコードする核酸。

[請求項8]
 請求項7に記載の核酸を発現可能に含むベクター。

[請求項9]
 請求項8に記載のベクターを含む形質転換細胞。

[請求項10]
 疾病細胞である、請求項9に記載の形質転換細胞。

[請求項11]
 請求項1~6のいずれか1項に記載のプローブ試薬、請求項8に記載のベクター、あるいは請求項9または10に記載の形質転換細胞を用いて、プロテアソーム活性を制御する候補物質の存在下で細胞におけるユビキチン-プロテアソーム系での該プローブ試薬蛋白質の分解活性を測定することを含む、ユビキチン-プロテアソーム系の異常に関連する疾病の治療剤をスクリーニングする方法。

[請求項12]
 プローブ試薬蛋白質の分解活性を、蛍光蛋白質IとIIの蛍光強度の比の変化として測定する、請求項11に記載の方法。

[請求項13]
 細胞がユビキチン-プロテアソーム系の異常に関連する疾病細胞である、請求項11または12に記載の方法。

[請求項14]
 請求項1~6のいずれか1項に記載のプローブ試薬または請求項8に記載のベクターと、疾病患者からの細胞または細胞抽出液とを接触させて該プローブ試薬蛋白質の分解活性を測定することを含む、ユビキチン-プロテアソーム系の異常と疾病との関連を調べる方法。

[請求項15]
 プローブ試薬内の分解を受ける蛋白質(デグロン蛋白質)が疾病に関連する蛋白質である、請求項11または14に記載の方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • RIKEN
  • Inventor
  • MIYAWAKI Atsushi
  • HIRANO Masahiko
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT RO RS RU SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
Reference ( R and D project ) ERATO MIYAWAKI Life Function Dynamics AREA
Please contact us by E-mail or facsimile if you have any interests on this patent.

PAGE TOP

close
close
close
close
close
close