Sexton, K.B., Witte, M.D., Blum, G. & Bogyo, M. Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain. Bioorg. Med. Chem. Lett. 17, 649-653

Leiden University, Leyden, South Holland, Netherlands
Bioorganic & Medicinal Chemistry Letters (Impact Factor: 2.42). 03/2007; 17(3):649-53. DOI: 10.1016/j.bmcl.2006.10.100
Source: PubMed


Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here, the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3, and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell-permeable reagents for monitoring legumain activity in complex proteomes.

Download full-text


Available from: Matthew Bogyo
  • Source
    • "Peptide AOMKs have been successfully used as covalent, irreversible probes of multiple classes of cysteine protease (Berger, et al., 2006; Bromme, et al., 1994; Sexton, et al., 2007). Furthermore, these compounds can be readily synthesized using solid phase synthesis methods (Kato, et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.
    Full-text · Article · Jun 2011 · Chemistry & biology
  • Source
    • "Aspartyl peptidyl flouromethyl and choloromethyl ketones have been reported to efficiently inhibit other cysteine proteases such as cathepsin B, S and V as well as legumain (Rozman-Pungercar et al., 2003). More recently, aspartyl peptidic acyloxymethyl ketones have been utilized as efficient cell-permeable activity-based probes for legumain (Sexton et al., 2007). Due to the aforementioned studies, all three inhibitors were screened against multiple cathepsins and legumain to evaluate cross-reactivity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Huntington's Disease (HD) is characterized by a mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats on the N terminus of the Htt protein. Numerous studies have identified Htt proteolysis as a critical pathological event in HD postmortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD therapeutic targets. We report the use of the substrate activity screening method against caspase-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models these irreversible inhibitors suppressed Hdh(111Q/111Q)-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study, the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development.
    Full-text · Article · Nov 2010 · Chemistry & biology
  • Source
    • "For cysteine peptidase labeling, an aliquot of the gut tissue extract (0.1–1.0 μg of protein) was incubated (1 h at 35 °C) with 1 μM of the active-site probe: Green-DCG-04 (Greenbaum et al., 2002), Fhex-PD-AOMK (Sexton et al., 2007), or FY01 (Yuan et al., 2006). The reaction was performed in 25 mM Na-acetate including 2.5 mM DTT and 25 mM NaCl (for cathepsin C) at pH 4.5 (for legumain) or pH 5.5 (for cathepsin B, L and C). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.
    Full-text · Article · Oct 2009 · Chemistry & biology
Show more