The neural cell adhesion molecules L1 and CHL1 are cleaved by BACE1 protease in vivo.

The ß-site amyloid precursor protein-cleaving enzyme BACE1 is a prime drug target for Alzheimer disease. However, the function and the physiological substrates of BACE1 remain largely unknown. In this work, we took a quantitative proteomic approach to analyze the secretome of primary neurons after acute BACE1 inhibition, and we identified several novel substrate candidates for BACE1. Many of these molecules are involved in neuronal network formation in the developing nervous system. We selected the adhesion molecules L1 and CHL1, which are crucial for axonal guidance and maintenance of neural circuits, for further validation as BACE1 substrates. Using both genetic BACE1 knock-out and acute pharmacological BACE1 inhibition in mice and cell cultures, we show that L1 and CHL1 are cleaved by BACE1 under physiological conditions. The BACE1 cleavage sites at the membrane-proximal regions of L1 (between Tyr(1086) and Glu(1087)) and CHL1 (between Gln(1061) and Asp(1062)) were determined by mass spectrometry. This work provides molecular insights into the function and the pathways in which BACE1 is involved, and it will help to predict or interpret possible side effects of BACE1 inhibitor drugs in current clinical trials.

Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1.

ß-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ∼0.76 nm), in primary neurons (EC(50) ∼1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ∼0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.

Evaluation of the type I signal peptidase as antibacterial target for biofilm-associated infections of Staphylococcus epidermidis.

The development of antibacterial resistance is inevitable and is a major concern in hospitals and communities. Moreover, biofilm-grown bacteria are less sensitive to antimicrobial treatment. In this respect, the Gram-positive Staphylococcus epidermidis is an important source of nosocomial biofilm-associated infections. In the search for new antibacterial therapies, the type I signal peptidase (SPase I) serves as a potential target for development of antibacterials with a novel mode of action. This enzyme cleaves off the signal peptide from secreted proteins, making it essential for protein secretion, and hence for bacterial cell viability. S. epidermidis encodes three putative SPases I (denoted Sip1, Sip2 and Sip3), of which Sip1 lacks the catalytic lysine. In this report, we investigated the active S. epidermidis SPases I in more detail. Sip2 and Sip3 were found to complement a temperature-sensitive Escherichia coli lepB mutant, demonstrating their in vivo functional activity. In vitro functional activity of purified Sip2 and Sip3 proteins and inhibition of their activity by the SPase I inhibitor arylomycin A(2) were further illustrated using a fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we demonstrated that SPase I not only is an attractive target for development of novel antibacterials against free-living bacteria, but also is a feasible target for biofilm-associated infections.

Substrate based peptide aldehyde inhibits bacterial type I signal peptidase.

Bacterial type I signal peptidase is a potential target for the development of novel antibacterial agents. In this study we demonstrate that a substrate based peptide aldehyde inhibits signal peptidases with a lower IC(50) value than the lipopeptides described to date. The length of the core lipopeptide could be reduced by removing several amino acids from both termini. Conversion of this peptide to an aldehyde resulted in a molecule with an IC(50) value of 0.09microM when tested against Staphylococcus [corrected] aureus SPase I, SpsB.

Enzymatic investigation of the Staphylococcus aureus type I signal peptidase SpsB - implications for the search for novel antibiotics.

Staphylococcus aureus has one essential type I signal peptidase (SPase), SpsB, which has emerged as a potential target in the search for antibiotics with a new mode of action. In this framework, the biochemical properties of SpsB are described and compared with other previously characterized SPases. Two different substrates have been used to assess the in vitro processing activity of SpsB: (a) a native preprotein substrate immunodominant staphylococcal antigen A and (b) an intramolecularly quenched fluorogenic synthetic peptide based on the sequence of the SceD preprotein of Staphylococcus epidermidis for fluorescence resonance energy transfer-based analysis. Activity testing at different pH showed that the enzyme has an optimum pH of approximately 8. The pH-rate profile revealed apparent pK(a) values of 6.6 and 8.7. Similar to the other SPases, SpsB undergoes self-cleavage and, although the catalytic serine is retained in the self-cleavage product, a very low residual enzymatic activity remained. In contrast, a truncated derivative of SpsB, which was nine amino acids longer at the N-terminus compared to the self-cleavage product, retained activity. The specificity constants (k(cat)/K(m)) of the full-length and the truncated derivative were 1.85 +/- 0.13 x 10(3) m(-1).s(-1) and 59.4 +/- 6.4 m(-1).s(-1), respectively, as determined using the fluorogenic synthetic peptide substrate. These observations highlight the importance of the amino acids in the transmembrane segment and also those preceding the catalytic serine in the sequence of SpsB. Interestingly, we also found that the activity of the truncated SpsB increased in the presence of a non-ionic detergent.

An easy and fast method for the evaluation of Staphylococcus epidermidis type I signal peptidase inhibitors.

In the framework of the search for new antimicrobial therapies to combat resistant bacteria, the type I signal peptidase (SPase I) serves as a potentially interesting target for the development of antibacterials with a new mode of action. Bacterial SPases I play a key role in protein secretion as they are responsible for the cleavage of signal peptides from secreted proteins. For the Gram-positive Staphylococcus epidermidis, an important source of biofilm-associated infections, three putative SPases I (denoted Sip1, Sip2, Sip3) have been described, of which Sip1 lacks the catalytic lysine. Here, we report the in vitro activity of purified Sip2 and Sip3 using pre-SceD as a native preprotein substrate of S. epidermidis and in a FRET-based assay. For the latter, a novel internally quenched fluorescent peptide substrate based on the signal peptide sequence of this native preprotein was developed and specific cleavage of this synthetic fluorogenic peptide substrate was demonstrated. The latter in vitro assay represents a rapid and reliable tool in future research for the identification and validation of potential SPase I inhibitors.