naa a22 4500 51074575X CHVBK 20180411083045.0 cr unu---uuuuu 180411e20130101xx s 000 0 eng 10.1007/s12192-012-0355-1 doi (NATIONALLICENCE)springer-10.1007/s12192-012-0355-1 Temperature-induced changes of HtrA2(Omi) protease activity and structure [Elektronische Daten] [Dorota Zurawa-Janicka, Miroslaw Jarzab, Agnieszka Polit, Joanna Skorko-Glonek, Adam Lesner, Agata Gitlin, Artur Gieldon, Jerzy Ciarkowski, Przemyslaw Glaza, Agnieszka Lubomska, Barbara Lipinska] HtrA2(Omi), belonging to the high-temperature requirement A (HtrA) family of stress proteins, is involved in the maintenance of mitochondrial homeostasis and in the stimulation of apoptosis, as well as in cancer and neurodegenerative disorders. The protein comprises a serine protease domain and a postsynaptic density of 95kDa, disk large, and zonula occludens 1 (PDZ) regulatory domain and functions both as a protease and a chaperone. Based on the crystal structure of the HtrA2 inactive trimer, it has been proposed that PDZ domains restrict substrate access to the protease domain and that during protease activation there is a significant conformational change at the PDZ-protease interface, which removes the inhibitory effect of PDZ from the active site. The crystal structure of the HtrA2 active form is not available yet. HtrA2 activity markedly increases with temperature. To understand the molecular basis of this increase in activity, we monitored the temperature-induced structural changes using a set of single-Trp HtrA2 mutants with Trps located at the PDZ-protease interface. The accessibility of each Trp to aqueous medium was assessed by fluorescence quenching, and these results, in combination with mean fluorescence lifetimes and wavelength emission maxima, indicate that upon an increase in temperature the HtrA2 structure relaxes, the PDZ-protease interface becomes more exposed to the solvent, and significant conformational changes involving both domains occur at and above 30°C. This conclusion correlates well with temperature-dependent changes of HtrA2 proteolytic activity and the effect of amino acid substitutions (V226K and R432L) located at the domain interface, on HtrA2 activity. Our results experimentally support the model of HtrA2 activation and provide an insight into the mechanism of temperature-induced changes in HtrA2 structure. The Author(s), 2012 HtrA2 human protease nationallicence Temperature dependence of HtrA2 activity nationallicence HtrA2 structural changes nationallicence Fluorescence spectroscopy nationallicence Acrylamide quenching nationallicence Trp mutants nationallicence Zurawa-Janicka Dorota Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut Jarzab Miroslaw Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut Polit Agnieszka Department of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland aut Skorko-Glonek Joanna Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut Lesner Adam Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut Gitlin Agata Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut Gieldon Artur Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut Ciarkowski Jerzy Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut Glaza Przemyslaw Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut Lubomska Agnieszka Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut Lipinska Barbara Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut Cell Stress and Chaperones Springer Netherlands 18/1(2013-01-01), 35-51 1355-8145 18:1<35 2013 18 12192 https://doi.org/10.1007/s12192-012-0355-1 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12192-012-0355-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Zurawa-Janicka Dorota Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Jarzab Miroslaw Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Polit Agnieszka Department of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland aut NATIONALLICENCE 700 1- Skorko-Glonek Joanna Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Lesner Adam Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Gitlin Agata Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Gieldon Artur Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Ciarkowski Jerzy Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Glaza Przemyslaw Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Lubomska Agnieszka Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut NATIONALLICENCE 700 1- Lipinska Barbara Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland aut NATIONALLICENCE 773 0- Cell Stress and Chaperones Springer Netherlands 18/1(2013-01-01), 35-51 1355-8145 18:1<35 2013 18 12192 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer