Two-hybrid analysis revealed that the C-terminal convertase P-domains of two proteases could interact with STI. These were an extracellular neutral zinc MTP encoded by SCO5447, and a probable serine protease encoded by SCO1355. Interaction between STI and SCO1355 was confirmed by in vitro analysis. It is therefore likely that these proteases are targets of STI, and although inhibition of their activity by STI has not yet been experimentally demonstrated, the inhibition of proteases by STI-type proteins is well established in the literatures (Takeuchi et al., 1992; Ueda et al., 1992; Kumazaki et al., 1993; Taguchi et al., 1998; Oda et al., 2001).
In eukaryote cells, proprotein convertase, containing a P-domain, is involved in the processing of precursors of many hormones, peptides and specific enzymes, including extracellular MTP (Ueda et al., 2003; Henrich et al., 2005). Often, such processing is important for the accurate regulation of cellular events (Rockwell et al., 2002). In yeast, convertase Kex2 generates mature α-mating factor and killer toxin from their precursors (Fuller et al., 1988). Kex2 and its mammalian analogues have a subtilisin-like catalytic domain and form a distinct subfamily (Kex2 endoproteinases) within the subtilisin superfamily (Nakayama, 1997). In mammalian cells, furin family proteases of the proprotein convertase type are also involved in tumour progression, disease and bacterial/viral infection (Bassi et al., 2005). In the mouse model, P-domain folding of proprotein convertase (PCSK5) was recently shown to be critical for its activity, and abnormal folding of the P-domain induced serious cellular defects in development, showing the importance of the P-domain in cellular processing (Szumska et al., 2008). Because of the importance of convertases, inhibitors of these enzymes have been studied as potential therapeutic agents for various diseases (Bontemps et al., 2007), and many synthetic convertase inhibitors have been evaluated (Basak, 2005). However, relatively few natural convertase inhibitors have been reported. In addition to inhibitors of mammalian convertase (furin) identified in humans (PI8) and Drosophila (Serpin4) (Dahlen et al., 1998; Osterwalder et al., 2004), another inhibitor, kexstatin I, which inhibits yeast kexin (Kex2), was found in the culture filtrate of Streptomyces platensis Q26. Kexstatin shows high similarity to STI (Oda et al., 2001), strongly supporting the general idea that the P-domain of proteases such as SCO5447 or SCO1355 might mark them for targeting by Streptomyces subtilisin inhibitor family protease inhibitors such as STI. In S. coelicolor, SCO5447 or SCO1355 proteases may process target proteins required for differentiation and Act production. Thus, in streptomycetes, as with yeast Kex2, a protease containing a P-domain may be involved in processing proteins needed for cellular events. In this context, it is interesting to note that SapB, a secreted morphogenetic peptide that is essential for aerial mycelium formation in streptomycetes under certain growth conditions, is derived from the ramS gene product by post-translational modification of specific amino acid residues, and proteolytic processing of a leader peptide sequence (Kodani et al., 2004). The ramC gene product is believed to encode the modification enzyme, while the protease has yet to be identified. Furthermore, in current work a screen for processing targets of SCO1355 protease has been undertaken using the exo-site scanning strategy developed for protease substrate screening (Overall et al., 1999; McQuibban et al., 2000). This has revealed a putative lipoprotein whose gene is closely linked to genes encoding morphogenetic proteins (rodlins and chaplins), suggesting a relationship between the protease cascade and morphogenesis (data not shown). These morphology-conferring proteins could potentially be the targets of the prokaryote convertase.
The somewhat similar colony phenotypes of the SCO1355 mutant and a STI-overproducing strain strengthen the idea that STI and SCO1355 protease are part of an extracellular cascade important for the formation of aerial mycelium. Inactivation of SCO5447, on the other hand, had no obvious morphological effects, possibly because of functional redundancy with the adjacent gene SCO5446. In addition to any involvement in processing of specific proprotein targets, the SCO5447 or SCO1355 proteases could also be involved in the reuse of vegetative biomass to generate nutrients supporting the growth of the aerial mycelium. Perhaps the increased death rate of stationary phase mycelium of a bldA mutant reflects a loss of control of the protease cascade.