GxGD\type intramembrane cleaving proteases (I\CLiPs) form a family of proteolytic enzymes that feature an aspartate\based catalytic mechanism. substrates. Both aspartates are localized on the cytoplasmic side of the membrane (Ng et?al. 2007; Hu et?al. 2011). Mutagenesis of these aspartates leads to the inactivation of FlaK (Bardy and Jarrell 2003). Accordingly, it is generally accepted that FlaK is an aspartic protease. FlaK does, however, not contain the classical D\T/S\G motif of prototypical aspartic proteases, but shows the conserved GxGD motif that is also found in TFPPs and presenilin (Steiner and Haass 2000). Also, the pH 10309-37-2 IC50 optimum of FlaK is in the neutral range, another similarity to the TFPPs (Bardy and Jarrell 2003). Based on these findings, it was suggested that FlaK and TFPPs might have 10309-37-2 IC50 a similar reaction mechanism and that both proteins are homologous concerning the structure of their active sites (Ng et?al. 2006). Because of the similarity to presenilin, both enzymes will also be often considered as model proteases for during the etiology of the neurodegenerative Alzheimer’s disease (AD) (Zhang et?al. 2012). Although a recent determination of the structure of was solved by protein crystallography (Hu et?al. 2011). It was shown that FlaK consists of two compactly folded domains, the JR1 has recently been solved. It does, however, also show an inactive conformation (Li et?al. 2013). The two catalytically important aspartates are herein separated by 6.7 ?, suggesting that substrate binding may result in a conformational switch. A more recent study from the same lab (Dang et?al. 2015) shows binding of a which laid the foundation to screen a small in\house library of structurally varied aspartic protease inhibitors. Our recognition of the 1st inhibitors of FlaK as well as their further chemical optimization and analysis resulted in the nonpeptidic compound 9, being an priceless tool to obtain deeper insights into the reaction mechanism of this family of aspartic proteases. Materials and Methods In vivo activity assay To analyze whether FlaK is definitely actively indicated in Tuner (Tuner (= 3.9?Hz), 124.7 (q, (%): 644 (100, [(%): 654 (100, [is actively indicated in TUNER (DE3), we co\indicated the enzyme with its substrate FlaB2. After induction with IPTG, both proteins should be indicated leading to a cleavage of the transmission peptide of FlaB2 by FlaK. Indeed, using western blot analysis against FlaB2, two bands representing the immature preflagellin FlaB2 and the adult flagellin FlaB2* were observed. In contrast, the manifestation of FlaB2 alone resulted in only one band of ~ 25?kDa (Fig.?1A). This demonstrates the heterologously indicated FlaK is definitely proteolytically active and excludes the presence of other peptidases capable 10309-37-2 IC50 of FlaB2 processing within the manifestation host. Based on this in 10309-37-2 IC50 vivo activity assay, we developed an in vitro assay with isolated and purified proteins. FlaK was solubilized from your membrane using the standard detergent DDM and purified to homogeneity by column chromatographic techniques. FlaB2, in contrast, could only become solubilized with the Mouse monoclonal to Alkaline Phosphatase denaturing detergent SDS. Therefore, FlaB2 was purified under denaturing conditions and its employment in the activity assay required the removal of SDS using acetone precipitation. Upon incubation with purified FlaK, this FlaB2 preparation was cleaved to FlaB2* resulting in the expected band with lower molecular excess weight upon analysis of the reaction by western blot (Fig.?1B). Interestingly, this reaction occurred in the detergent solubilized state without the addition of any lipid, showing that no membrane is required for the proteolytic reaction. Unfortunately, we did not observe complete conversion of FlaB2 to FlaB2* upon prolonged treatment with active FlaK (data not demonstrated), indicating that only about 50% of the substrate is in a state or conformation that can be processed or that FlaK is definitely inhibited by the product FlaB2*. However, this 10309-37-2 IC50 band shift assay is highly useful and is the basis for the development of specific FlaK inhibitors. Open in a separate window Number 1 Activity.