monash_80104.pdf (3.93 MB)
Distribution and functions of protease inhibitor 8 (SERPINB8)
thesisposted on 2017-02-03, 03:54 authored by Gillard, Anneliese
Protease inhibitor 8 (PI8) / SERPINB8 is an intracellular serpin, a member of a class of protease inhibitors which irreversibly inhibit serine proteases. Other serpins within clade B are also expressed intracellularly but are not secreted, and are usually found in an overlapping distribution pattern with their target protease. Some members of this clade have accepted roles in inhibition of cytotoxic granule proteases. The reactive centre loop (RCL) of PI8, which directs its specifi city towards a target protease, contains two putative proprotein convertase recognition sites, suggesting it may play a physiological role in the inhibition of one or more proprotein convertases. Furin and other mammalian endopeptidases of the proprotein convertase (PC) family are noted for their specifi city to the amino acid sequence Arg-X-X-(Lys/Arg). Such cleavage is a required step for the activation of numerous proteins involved in normal physiological processes as well as forming the basis of infectivity of viral coat proteins and bacterial toxins, and progression of tumour growth and arthritis. While the activity of PCs has been reported to be controlled at the transcription level, there is also evidence that inhibitory proteins exist which control the action of PCs in vivo. Several members of the serpin family have reportedly demonstrated in vitro activity against various PCs, most prominently, the engineered serpin α1-antitrypsin Portland, which has been postulated as a therapeutic agent (Tsuji et al, 2007). In mammals there is an endogenous serpin, PI8, which demonstrates rapid inhibition of furin, kinetically characteristic of a physiological inhibitory interaction (Dahlen et al, 1998). In this study the murine tissue mRNA distribution of members of the PC family was correlated with expression of PI8: furin, PC5/6, PACE4 and PC7 showed distribution overlapping with, but not identical to PI8. PI8 expression was also examined in other organs, and was found to be predominantly expressed within epithelial cell types. In a mouse model, mPI8 was shown to be upregulated in the epithelial-mesenchymal transition process in a model of kidney fi brosis. Further, this study demonstrated that recombinant mouse PI8 is able to inhibit the activity of several members of the PC family against the fl uorogenic substrate Boc-RVRR-AMC. Determination of the stoichiometry of inhibition for the interaction showed PI8 ineffi ciently inhibited PACE4, but is a better inhibitor of PC5/6b, and inhibited furin approximately at the physiological ratio of 1:1. This result provides support for the role of PI8 as a physiological inhibitor of furin, and is a novel fi nding of anti-proteolytic activity by PI8 against PACE4 and PC5/6b, suggesting PI8 may be involved in the physiological regulation of several prohormone convertases.