ES2579995T3 - Modulando la actividad de triglicérido hidrolasa - Google Patents

Modulando la actividad de triglicérido hidrolasa Download PDF

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Publication number
ES2579995T3
ES2579995T3 ES05745594.1T ES05745594T ES2579995T3 ES 2579995 T3 ES2579995 T3 ES 2579995T3 ES 05745594 T ES05745594 T ES 05745594T ES 2579995 T3 ES2579995 T3 ES 2579995T3
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Prior art keywords
activity
hydrolase activity
atgl
triglyceride hydrolase
cgi
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Active
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ES05745594.1T
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English (en)
Inventor
Rudolf Zechner
Robert Zimmermann
Juliane G. Strauss
Günter HÄMMERLE
Achim Lass
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Karl-Franzens-Universitaet Graz
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Karl-Franzens-Universitaet Graz
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procedimiento para determinar la actividad triglicérido hidrolasa de una proteína que comprende una cadena polipeptídica codificada por la secuencia de ADN de acuerdo con la SEQ NO. 1 en una muestra acuosa en presencia de lipasa sensible a hormonas, caracterizado por que se añade halogenuro de metal alcalino a la muestra en una cantidad eficaz para suprimir la actividad de dicha lipasa sensible a hormonas, después de lo que se determina la actividad triglicérido hidrolasa.

Description

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actividad de HSL (-68 % con KCl 1 M). En contraste, la actividad de ATGL fue estimulada por KCl (+ 84 % con KC1l 1 M.
Figura 6. CGI-58 activa específicamente la actividad de TGH de ATGL. Se clonaron ATGL, HSL, y CGI-58murinas en un vector de expresión pcDNA4/HisMax con marca His y las proteínas recombinantes se expresaron 5 temporalmente en células COS-7. Se utilizó -galactosidasa (LacZ) como control, (a) se detectaron las proteínas marcadas con His en los extractos citoplasmáticos de las células transfectadas mediante bandas Western usando un anticuerpo anti-His monoclonal, (b) se determinó la actividad de TGH de extractos citoplasmáticos de células transfectadas utilizando un sustrato de trioleína radiomarcada, (c) se mezclaron los extractos citoplasmáticos de células que expresan ATGL o HSL con extractos que contienen ya sea CGI-58 o LacZ y se determinó la actividad de 10 TGH. Se usó LacZ como control, (d) efecto dependiente de la dosis de CGI-58 sobre la actividad de TGH de ATGL. Se mezclaron extractos citoplasmáticos de células que expresan ATGL con concentraciones crecientes de extractos que expresan CGI-58 y se analizó la actividad de TGH. Los niveles de expresión de ATGL y CGI-58 en extractos citoplasmáticos se visualizaron mediante bandas Western utilizando un anticuerpo anti-His y se cuantificaron densitométricamente. Las proporciones molares se calcularon mediante el ajuste de la intensidad de la expresión de 15 la proteína recombinante respectiva marcada con His, (e) la activación de ATGL se analizó por la unión del inhibidor de lipasas fluorescente NBD-snlTG. Se incubaron extractos citoplasmáticos con inhibidor marcado con fluorescencia y se corrieron en un gel SDS-PAGE. Las proteínas marcadas con NBD-snlTG se visualizaron en un BioRad FX Pro Laserscanner. Los datos de los ensayos de actividad de TGH se presentan como media ± D.T. y representan al menos tres experimentos independientes. *p<0,05, **p<0,01, ***p<0,001 (f) Efecto dependiente de la dosis de hCGI
20 58 sobre la actividad de TGH de hATGL. Se determinó la proporción molar ATGL/CGI-58 como se describe en (d).
La Fig. 6 muestra que CGI-58 afecta el metabolismo de los lípidos como activador de ATGL y parece ser clave en el metabolismo lipídico celular. Con respecto a los altos niveles de expresión de CGI-58 y ATGL en el tejido adiposo, la modulación de la actividad de cada proteína podría afectar al metabolismo de lo TG y los AGL y por lo tanto ofrecer una estrategia para el tratamiento de la obesidad y de los trastornos relacionados.
25 Listado de referencias
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Claims (1)

  1. imagen1
ES05745594.1T 2004-05-27 2005-05-27 Modulando la actividad de triglicérido hidrolasa Active ES2579995T3 (es)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT9242004 2004-05-27
AT9242004 2004-05-27
PCT/EP2005/005710 WO2005115461A2 (en) 2004-05-27 2005-05-27 Pharmaceutical composition for modulating the activity of triglyceride hydrolase

Publications (1)

Publication Number Publication Date
ES2579995T3 true ES2579995T3 (es) 2016-08-18

Family

ID=34968673

Family Applications (1)

Application Number Title Priority Date Filing Date
ES05745594.1T Active ES2579995T3 (es) 2004-05-27 2005-05-27 Modulando la actividad de triglicérido hidrolasa

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Country Link
US (2) US20090018066A1 (es)
EP (1) EP1765997B1 (es)
ES (1) ES2579995T3 (es)
WO (1) WO2005115461A2 (es)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2921270B1 (fr) * 2007-09-25 2012-08-10 Limousine D Applic Biolog Ditesilab Soc Ind Utilisation d'actifs agissant sur les proteines atgl et/ou cgi-58.
WO2010115825A2 (en) * 2009-03-31 2010-10-14 Robert Zimmermann Modulation of adipose triglyceride lipase for prevention and treatment of cachexia, loss of weight and muscle atrophy and methods of screening therefor
WO2010112569A1 (en) * 2009-03-31 2010-10-07 Robert Zimmermann Modulation of adipose triglyceride lipase for prevention and treatment of cachexia, loss of weight and muscle atrophy and methods of screening therefor
US20140147431A1 (en) * 2010-09-20 2014-05-29 The Regents Of The University Of California Compositions and Methods for Modulating Desnutrin-Mediated Adipocyte Lipolysis
EP2956162B1 (en) * 2013-02-15 2019-05-15 Ruprecht-Karls-Universität Heidelberg Abhd5 for use in the treatment of heart failure

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2380317A1 (en) * 1999-07-22 2001-02-01 Incyte Genomics, Inc. Human synthetases
WO2005014645A2 (en) * 2003-08-07 2005-02-17 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Pedf-r receptor and uses
US7473541B2 (en) * 2004-07-09 2009-01-06 Gross Richard W Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities

Also Published As

Publication number Publication date
US20120009613A1 (en) 2012-01-12
WO2005115461A3 (en) 2006-05-04
EP1765997B1 (en) 2016-04-13
WO2005115461A2 (en) 2005-12-08
US20090018066A1 (en) 2009-01-15
EP1765997A2 (en) 2007-03-28

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