Computational Systems Biochemistry Group

Proteasomes are cellular, multicatalytic protease complexes that degrade intracellular proteins into smaller peptides. The 20S proteasome (700 kDa) is a cylindrical particle consisting of 28 subunits (molecular masses 21-35 kDa) arranged in four stacked hepatmeric rings [1, 2]. Two β-rings form the central cavity of the cylinder and harbour the proteolytic acive sites at their inner surface. Unlike bacterial proteasomes 3, eukaryotic 20S proteasome possesses only three catalytically active β-subunits (β1, β2 and β5), which belong to the family of N-terminal nucleophile hydrolases [4, 5]. The α-subunits of the two outer rings form the boundary of a gated channel, through which the traffic of incoming substrates and outgoing peptides is likely to proceed [6, 7].

Proteasomes are involved in many regulatory and metabolic processes [8, 9, 10]. It has been shown that the proteasome itself can participate in the extraction of an ER-membrane protein from the lipid bilayer. Nevertheless, mechanistic details of how the proteasome may attack and digest immobilized substrates are largely unknown.

Abbau-Schema The aim of this study is to develop an in vitro assay to study the degradation of oligomeric peptide substrates that are immobilized by covalent attachment to various types of solid surfaces. We are using a pp89-derived 25-meric peptide from cytomegalo virus (Kloe411) in the solid-phase and the 20S proteasome isolated from erythrocytes. Proteolytic fragments are identified by mass spectroscopy. The fragment pattern obtained with the immobilized substrate is being compared with cleavage patterns generated with the soluble one. Finally, we should be able to generate a kinetic model from the fragment pattern.

Researchers

Marc Hovestädt
Prof. Hermann-Georg Holzhütter
in cooperation with the Molecular Libraries and Recognition Group, Dr. Rudolf Volkmer, Charité - Universitätsmedizin Berlin, Institute for Medical Immunology and Ulrike Kuckelkorn from Prof. Kloetzels group, Charité - Universitätsmedizin Berlin, Institute of Biochemistry

References

[1] Frank R. (1992) Spot synthesis an easy technique for positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48(42), 9217-9232 [Abstract]
[2] Wenschuh et al. (2000) Positionally adressable parallel synthesis on continuous membranes. Combinatorial Chemistry, A Practical Approach, Oxford University Press. ISBN 0 19 963754 7.
[3] Reineke, U., et al., Applications of peptide arrays prepared by the SPOT-technology. Curr Opin Biotechnol 12, 59-64 (2001).
[4] Bhargava, S., et al., A complete substitutional analysis of VIP for better tumor imaging properties. J Mol Recognit 15, 145-153 (2002).
[5] Kamradt, T., et al.., Cross-reactivity of T lymphocytes in infection and autoimmunity. Mol. Div. 8, 271-280, (2004)
[6] Paston, S. J. et al., Hum Immunol, 65, 544-9, (2004).
[7] Elkington, R. et al., Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 2003, 77, 5226-40 -
[8] Diamond, D. J. et al., Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 1997, 90, 1751-67
[9] Ay, B.; Streitz, M.; Boisguerin, P.; Schlosser, A.; Mahrenholz, C. C.; Schuck, S. D.; Kern, F.; Volkmer, R., Sorting and pooling strategy: A novel tool to map a virus proteome for CD8 T-cell epitopes. Biopolymers 2007, 88, (1), 64-75.
[10] Ay, B.; Volkmer, R.; Boisguerin, P., Synthesis of cleavable peptides with authentic C-termini: an application for fully automated SPOT synthesis. Tetrahedron Letters 2007, 48, (3), 361-364

		Analysis of Degradation of Support Fixed Peptides by the 20S Proteasome
Home Research VirtualLiver People Publications Software Positions Events Contact		Proteasomes are cellular, multicatalytic protease complexes that degrade intracellular proteins into smaller peptides. The 20S proteasome (700 kDa) is a cylindrical particle consisting of 28 subunits (molecular masses 21-35 kDa) arranged in four stacked hepatmeric rings [1, 2]. Two β-rings form the central cavity of the cylinder and harbour the proteolytic acive sites at their inner surface. Unlike bacterial proteasomes 3, eukaryotic 20S proteasome possesses only three catalytically active β-subunits (β1, β2 and β5), which belong to the family of N-terminal nucleophile hydrolases [4, 5]. The α-subunits of the two outer rings form the boundary of a gated channel, through which the traffic of incoming substrates and outgoing peptides is likely to proceed [6, 7]. Proteasomes are involved in many regulatory and metabolic processes [8, 9, 10]. It has been shown that the proteasome itself can participate in the extraction of an ER-membrane protein from the lipid bilayer. Nevertheless, mechanistic details of how the proteasome may attack and digest immobilized substrates are largely unknown. The aim of this study is to develop an in vitro assay to study the degradation of oligomeric peptide substrates that are immobilized by covalent attachment to various types of solid surfaces. We are using a pp89-derived 25-meric peptide from cytomegalo virus (Kloe411) in the solid-phase and the 20S proteasome isolated from erythrocytes. Proteolytic fragments are identified by mass spectroscopy. The fragment pattern obtained with the immobilized substrate is being compared with cleavage patterns generated with the soluble one. Finally, we should be able to generate a kinetic model from the fragment pattern. Researchers Marc Hovestädt Prof. Hermann-Georg Holzhütter in cooperation with the Molecular Libraries and Recognition Group, Dr. Rudolf Volkmer, Charité - Universitätsmedizin Berlin, Institute for Medical Immunology and Ulrike Kuckelkorn from Prof. Kloetzels group, Charité - Universitätsmedizin Berlin, Institute of Biochemistry References [1] Frank R. (1992) Spot synthesis an easy technique for positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48(42), 9217-9232 [Abstract] [2] Wenschuh et al. (2000) Positionally adressable parallel synthesis on continuous membranes. Combinatorial Chemistry, A Practical Approach, Oxford University Press. ISBN 0 19 963754 7. [3] Reineke, U., et al., Applications of peptide arrays prepared by the SPOT-technology. Curr Opin Biotechnol 12, 59-64 (2001). [4] Bhargava, S., et al., A complete substitutional analysis of VIP for better tumor imaging properties. J Mol Recognit 15, 145-153 (2002). [5] Kamradt, T., et al.., Cross-reactivity of T lymphocytes in infection and autoimmunity. Mol. Div. 8, 271-280, (2004) [6] Paston, S. J. et al., Hum Immunol, 65, 544-9, (2004). [7] Elkington, R. et al., Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 2003, 77, 5226-40 - [8] Diamond, D. J. et al., Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 1997, 90, 1751-67 [9] Ay, B.; Streitz, M.; Boisguerin, P.; Schlosser, A.; Mahrenholz, C. C.; Schuck, S. D.; Kern, F.; Volkmer, R., Sorting and pooling strategy: A novel tool to map a virus proteome for CD8 T-cell epitopes. Biopolymers 2007, 88, (1), 64-75. [10] Ay, B.; Volkmer, R.; Boisguerin, P., Synthesis of cleavable peptides with authentic C-termini: an application for fully automated SPOT synthesis. Tetrahedron Letters 2007, 48, (3), 361-364