Trypanosoma brucei: β2-selective proteasome inhibitors do not block the proteasomal trypsin-like activity but are trypanocidal

Previous studies indicated that the proteasome of the protozoan parasite Trypanosoma brucei is particularly sensitive to inhibition of the trypsin-like activity. In this study, three newly developed β2 subunit-specific inhibitor (LU-102, LU-002c and LU-002i) were tested for their ability to block the trypsin-like activity of the trypanosomal proteasome. At 10 µM, none of the compounds affected the proteasomal trypsin-like activity in cell lysates of bloodstream forms of T. brucei. On the other hand, leupeptin, a well-established β2 inhibitor, supressed the proteasomal trypsin-like activity within trypanosome cell lysates with a 50% inhibitory concentration of 2 µM demonstrating the inhibitability of the trypsin-like activity of the T. brucei proteasome under the experimental condition. Nevertheless, two compounds (LU-102 and LU-002i) displayed moderate trypanocidal activity with 50% growth inhibition values of 6.9 and 8.5 µM, respectively. In the case of LU-102, it was shown that the trypanocidal activity of the compound was due to inhibition of the major lysosomal cysteine protease TbCATL. The main finding of this study indicate substantial inhibitor sensitivity differences between the trypsin-like sites of the human and trypanosomal proteasomes. Whether these differences can be exploited for the design of anti-trypanosomal drug therapies remains to be shown

Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology

In this review, we will discuss the enzymes that are involved in the synthesis and degradation of glycoconjugates and we will give an overview of the inhibitors and activity-based probes (ABPs) that have been used to study these. Following discussion of some general aspects of the biosynthesis and degradation of N-linked glycoproteins, attention is focused on the enzymes that hydrolyze the protein–carbohydrate linkage, peptide N-glycanase and glycosylasparaginase and their mechanism. We then focus on the biosynthesis of O-linked glycoproteins and glycolipids and in particular on the enzymes that hydrolyze the interglycosidic linkages in these, the glycosidases. Some important mechanism-based glycosidase inhibitors that form a covalent bond with the targeted enzyme(s), their corresponding ABPs and their application to study this class of enzymes are highlighted. Finally, alternative pathways for degradation of glycoconjugates and an ABP-based strategy to study these will be discussed

Trypanosoma brucei: inhibition of cathepsin L is sufficient to kill bloodstream forms

The lysosomal cysteine protease activity of Trypanosoma brucei comprises a cathepsin B enzyme (TbCATB) and a cathepsin L enzyme (TbCATL). Inhibition of the cysteine protease activity is lethal to bloodstream-form trypanosomes but it was not entirely clear which of the two enzymes are essential for survival of the parasites. Here we show that the vinyl sulfone compound LU-102 selectively inhibits TbCATL without affecting TbCATB and the proteasomal trypsin-like activity within trypanosomes. Therefore, the trypanocidal activity displayed by LU-102 can be attributed solely to the inhibition of TbCATL demonstrating that this enzyme is essential to the survival of T. brucei

High Immunoproteasome Activity and sXBP1 in Pediatric Precursor B-ALL Predicts Sensitivity towards Proteasome Inhibitors

Proteasome inhibitors (PIs) are approved backbone treatments in multiple myeloma. More recently, inhibition of proteasome activity with the PI bortezomib has been clinically evaluated as a novel treatment strategy in pediatric acute lymphoblastic leukemia (ALL). However, we lack a marker that could identify ALL patients responding to PI-based therapy. By using a set of activity-based proteasome probes in conjunction with cytotoxicity assays, we show that B-cell precursor ALL (BCP-ALL), in contrast to T-ALL, demonstrates an increased activity of immunoproteasome over constitutive proteasome, which correlates with high ex vivo sensitivity to the PIs bortezomib and ixazomib. The novel selective PI LU015i-targeting immunoproteasome β5i induces cytotoxicity in BCP-ALL containing high β5i activity, confirming immunoproteasome activity as a novel therapeutic target in BCP-ALL. At the same time, cotreatment with β2-selective proteasome inhibitors can sensitize T-ALL to currently available PIs, as well as to β5i selective PI. In addition, levels of total and spliced forms of XBP1 differ between BCP-ALL and T-ALL, and only in BCP-ALL does high-spliced XBP1 correlate with sensitivity to bortezomib. Thus, in BCP-ALL, high immunoproteasome activity may serve as a predictive marker for PI-based treatment options, potentially combined with XBP1 analyses

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic β-subunits

AbstractBackground: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, β1, β2 and β5, which are replaced in the γ-interferon-inducible immunoproteasome by a different set of catalytic subunits, β1i, β2i and β5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as ‘chymotryptic-like’ (β5), ‘tryptic-like’ (β2) and ‘peptidyl-glutamyl peptide hydrolyzing’ (β1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits.Results: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulfone (AdaAhx3L3VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels.Conclusions: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts

Analysis of Protease Activity in Live Antigen-presenting Cells Shows Regulation of the Phagosomal Proteolytic Contents During Dendritic Cell Activation

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow–derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte

Bacteroides fragilis fucosidases facilitate growth and invasion of Campylobacter jejuni in the presence of mucins

The enteropathogenic bacterium Campylobacter jejuni was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilization. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains 129,108 and NCTC 11168 increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. C. jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion. This article is protected by copyright. All rights reserved