Abstract
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 95 kDa, 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-012-0355-1) contains supplementary material, which is available to authorized users.
Highlights
It is of biological importance that the hydrolytic enzymes catalyzing protein degradation should exhibit their activity at the proper cellular or extracellular location and under specific physiological conditions only
We found that HtrA2 activity was highly dependent on temperature: it was practically negligible at 20 °C and increased about 14fold between 25 and 45 °C (Fig. 1a)
The marked increase of HtrA2 activity in conjunction with rising temperatures is consistent with its main physiological role, which is to serve as a protein control guard and maintain mitochondrial homeostasis
Summary
It is of biological importance that the hydrolytic enzymes catalyzing protein degradation should exhibit their activity at the proper cellular or extracellular location and under specific physiological conditions only. These enzymes remain in inactive forms until their function is required. This rule applies to the high-temperature requirement A (HtrA) family of proteins whose members are very well conserved in evolution. They have proteolytic and chaperone activity and recognize unfolded proteins with exposed hydrophobic stretches. Four human members of this family, HtrA1–HtrA4, have been identified, and it has been shown that they participate in protein quality control, regulation of cell proliferation, cell migration and fate (recently reviewed by Clausen et al 2011; Singh et al 2011)
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