Proceedings book download - 5Z.com

solved using molecular replacement technique where proteasome (3RYP) structure as amodel was used (refinement still in progress).Activity of designed peptides and digestion experimentSynthesized compounds are based on the sequences of 11S activator, protein HIV-1 Tatand PR39 peptide respectively. Synthesis was carried out on solid phase according to SPPSprocedure on automatic peptides synthesizer Millipore 9050 Plus Pepsynthesizer ormicrowave reactor Plazmatronika RM800. Biological studies shown that followingpeptides are able to activate latent proteasome with high effectiveness (data not shown).Even if peptides are able to regulate proteasome activity, there is a need to check theirdegradation by multicatalytic complex. To determine stability of mentioned compounds,digestion experiment was performed as follows: peptides concentration used for experiment- 100μM, proteasome concentration 0.01 mg/ml, reaction buffer: 50 mM Tris-Cl pH 8.0,0.005% SDS. Results are shown on MALDI-MS spectra (Figure 2).Fig. 2. MALDI MS spectra of digestion experiment and procedure of experiment.ConclusionsSince precise regulation of proteasome functioning plays very important role in keepingorganisms in homeostasis, there is a need to find selective and specific inhibitors oractivators of its activity. High level of activity and allosteric character of synthesizedpeptides gave the idea to crystallize the protein itself and in complex with those regulators.That allows us to compare conformational changes between proteasome in native state andinterrupted state caused by interaction with peptide molecule (crystallization of proteincomplexes in progress). Digestion experiment indicated that used compounds were notdigested by enzyme. To make conditions most suitable for substrate degradation, activatedproteasome was used. Even after 4h of incubation there were no traces of any othercompound except used peptide – MS spectra above. Comparison of structures of proteincomplex and protein itself should give an answer about mechanism of regulator-proteasomeinteraction.AcknowledgmentsSupported by grant DS/8440-4-0172-0.References1. Juryszyn, A., Skotnicki, A. Adv. Clin. Exp. Med. 15, 309-320 (2006).2. Rechsteiner, M., Realini, C., Ulster V. Biochem. J. 345, 1-15 (2000).3. Ciechanover, A., Iwai, K. IUBMB Life 56, 193-201 (2004).4. Debirage, R., Price, R. Am. J. Physiol. Renal Physiol. 285, F1-8 (2003).497