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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Search for Inhibitors or Activators of Human ProteasomeR. Rostankowski 1,2 , E. Jankowska 1 , M. Gaczyńska 3 , P.A. Osmulski 3 ,S. Madabhushi 3 , and F. Kasprzykowski 11 Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952, Gdansk, Poland;2 Department of Biochemistry, University of Texas Southwestern Medical Center, 5323Harry Hines Blvd, Dallas, 75309, TX, U.S.A.; 3 Institute of Biotechnology, University ofTexas Health Science Center, 15355 Lambda Dr, San Antonio, 78245, TX, U.S.A.IntroductionCell metabolism is strictly dependent on number and activity of many different proteins,which are regulated by transcription, translation and post-translative modification, as wellas their irreversible proteolysis effectivity. One of the degradation systems, present ineukaryotic cells, is ATP-dependent ubiquitin-proteasome pathway [1]. Proteasome ismulticatalytic protein complex responsible for intracellular protein degradation. Theproteasome is made up of two subcomplexes: a catalytic core particle (CP; also known asthe 20S proteasome) and one or two terminal 19S regulatory particles (RP) that serves as aproteasome activator with a molecular mass of approximately 700 kDa (called PA700). The19S RP binds to one or both ends of the latent 20S proteasome to form an enzymaticallyactive proteasome 26S. The eukaryotic proteasome is a multicatalytic proteasecharacterized by three activities with distinct specificities against short synthetic peptides: a“chymotryptic-like” activity with preference for tyrosine or phenylalanine at the P1position; a “tryptic-like”activity with preference for arginine or lysine at the P1 position;and a “postglutamyl” hydrolyzing activity with a preference for glutamate or other acidicresidues at the P1 position. [2]. Proteasome activity has a major role in homeostasis of thewhole organism. Disturbance of regular functioning of proteasome is observed in manydiseases, for example neurodegenerative illnesses such as Parkinson, Alzheimer orHuntington [3]. An increase of proteasome activity is also observed in case ofimmunological and pulmonary illnesses like mucoviscidosis [4]. Due to its functionnowadays researchers of the world try to find new regulators of proteasome activity.Results and DiscussionYeast culturing: Saccharomyces cerev, strain MHY501 (kindly provided by MarkHochstrasser, Yale University) was inoculated in YPD medium (yeast extract, peptone,D-Glucose). After culturing, yeast cells wereharvested and processed using microfluidizer andhomogeniztion grinder respectively. Isolated proteinwas purified in three steps: anion exchangechromatography, hydroxyapatite chromatographyand gel filtration as a last step.Purification: Anion exchange chromatographypurification used following protocol: equilibration ofcolumn, direct load, column buffer, 250mM NaCl,400mM NaCl, 1M NaCl with 5-10 min at 4 o C, 500rpm spinning in every stepFig. 1. Proteasome crystals.Hydroxyapatite purification: Purification usedfollowing protocol: 10mM sodium phosphate buffer,100mM, 200mM, 300mM, 400mM and 500mMbuffer with 5 min at 4 o C , 200 g spinning in every step.Gel filtration: Purification proceeded as follows: isocratic separation against gel filtrationbuffer containing: 50 mM Tris-Cl pH 7.0 and 10% glycerol on Superose 6 column. Flowrate 0.4 ml/min and 0.5 ml of fraction volume.Crystallization: Hanging drop vapor diffusion technique was used for the crystallization ofprotesome. The best diffracting crystals (Figure 1) were obtained under followingconditions: 0.03 M Magnessium acetate, 0.1 M MES 7.2, 12% (v/v) MPD. Proteinconcentration used for experiment was 2.5 mg/ml and data were collected using advancesphoton source – beam 19ID (Argonne National Laboratory, Argonne, IL). Structure was496