Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides

Cezari, Maria Helena S.; Puzer, Luciano; Juliano, María A.; Carmona, Adriana K.; Juliano, Luiz

doi:10.1042/bj20020840

Cited by 37 publications

(42 citation statements)

References 31 publications

(33 reference statements)

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“…Therefore we used CatB (11.6 nM) and CatL (13 nM) concentrations that were approximately 10 times higher compared with CatS (1.3 nM) in these assays. Another difficulty in creating a CatS-specific substrate was that CatB as well as CatL show a peptidyl-dipeptidase activity of relatively broad specificity, allowing C-terminal truncations of dipeptides (7,8). To block this activity, substrates contained a protecting D-Arg residue at the C terminus.…”

Section: Resultsmentioning

confidence: 99%

“…Although CatS is one of the major proteases involved in antigen processing (4 -6), specific determination of its activity in antigen presenting cells is currently complicated because a specific substrate is not yet known. One reason for this is that CatB, CatL, and CatS show relatively similar endopeptidase specificities, and CatB and CatL possess a peptidyl-dipeptidase activity of relatively broad specificity as well (7)(8)(9). The development of a specific substrate for CatS should be useful to illuminate the complexity of the class II MHC-restricted pathway of antigen presentation, and knowledge of the protease specificity of enzymes involved in antigen processing, for example that of CatS, could help to create software for antigenic peptide prediction.…”

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confidence: 99%

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Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate

Lützner

Kalbacher

2008

Journal of Biological Chemistry

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Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGL-RWE-Lys(Dnp)-DArg-NH 2 , which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3 was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1, and the P-3 substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPP-MGLPWE-Lys(Dnp)-DArg-NH 2 and Mca-GRWHPMGAPWELys(Dnp)-DArg-NH 2 , did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.Lysosomal cysteine proteases of the papain family were long believed to be exclusively involved in nonspecific proteolysis within the lysosome. However, the use of specific protease inhibitors and the study of gene knock-out mice suggested that some of them are involved in many other processes, such as cellular homeostasis, autophagy, apoptosis, and antigen presentation (1-3). Antigen presentation by MHC 2 class II molecules requires the entry of antigens into the endosomal-lysosomal compartment. These antigens are then processed by proteolytic enzymes, of which the lysosomal cysteine proteases of the papain family constitute an important subset. The generated peptides bind to MHC class II molecules, which are then displayed at the surface of professional APCs including macrophages, dendritic cells (DCs), and B cells. The variety of MHC class II peptide products that can activate CD4 ϩ T cells is on the one hand determined by allelic variation in MHC molecule binding specificity and on the other by the identity and level of activity of processing proteases present in APCs. Although CatS is one of the major proteases involved in antigen processing (4 -6), specific determination of its activity in antigen presenting cells is currently complicated because a specific substrate is not yet known. One reason for this is that CatB, CatL, and CatS show relati...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate

Lützner

Kalbacher

2008

Journal of Biological Chemistry

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“…The supplemental Table S5 compares kinetic parameters of the mode-selective substrates. The endopeptidase substrates of cathepsins B (59), Abz-Gln-Val-ValAla-Gly-Ala-EDDnp, and Abz-Ala-Phe-Arg-Phe-Ser-GlnEDDnp, displayed 2 orders of magnitude lower k cat /K m values than the carboxydipeptidase substrates Abz-Phe-Arg-ValNph and Abz-Phe-Arg-Nph-Val (60,61). With the minimized endopeptidase substrate, Cbz-Phe-Arg-AMC (62), k cat /K m for SmCB1 was 1 order of magnitude lower than for the carboxydipeptidase substrates.…”

Section: Determination Of Crystal Structures-recombinantmentioning

confidence: 99%

“…the primed substrate-binding subsites of SmCB1. The structure of the synthesized libraries Abz-Phe-Arg-Xaa-Nph-OH and Abz-Phe-Arg-Nph-Xaa-OH contain two fixed residues, PheArg in P2-P1 that are favored by SmCB1 and other cathepsins B (60,61,63), and help to anchor the substrates in nonprimed subsites. The substitutions in the Xaa positions define the P1Ј and P2Ј residues.…”

Section: Table 1 Inhibition Of Smcb1 By Vinyl Sulfone Inhibitors and mentioning

confidence: 99%

Structural Basis for Inhibition of Cathepsin B Drug Target from the Human Blood Fluke, Schistosoma mansoni

Jílková

Řezáčová

Lepšı́k

et al. 2011

Journal of Biological Chemistry

115

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Schistosomiasis caused by a parasitic blood fluke of the genus Schistosoma afflicts over 200 million people worldwide. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated peptidase that digests host blood proteins as a source of nutrients. It is under investigation as a drug target. To further this goal, we report three crystal structures of SmCB1 complexed with peptidomimetic inhibitors as follows: the epoxide CA074 at 1.3 Å resolution and the vinyl sulfones K11017 and K11777 at 1.8 and 2.5 Å resolutions, respectively. Interactions of the inhibitors with the subsites of the active-site cleft were evaluated by quantum chemical calculations. These data and inhibition profiling with a panel of vinyl sulfone derivatives identify key binding interactions and provide insight into the specificity of SmCB1 inhibition. Furthermore, hydrolysis profiling of SmCB1 using synthetic peptides and the natural substrate hemoglobin revealed that carboxydipeptidase activity predominates over endopeptidolysis, thereby demonstrating the contribution of the occluding loop that restricts access to the active-site cleft. Critically, the severity of phenotypes induced in the parasite by vinyl sulfone inhibitors correlated with enzyme inhibition, providing support that SmCB1 is a valuable drug target. The present structure and inhibitor interaction data provide a footing for the rational design of anti-schistosomal inhibitors.

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“…These enzymes are endoproteases, in contrast to exoproteases, which are further divided as the aminopeptidase CatH (also shows endoprotease activity), the carboxypeptidases CatA and CatX and the peptidyl dipeptidase CatB. The exoprotease CatB releases two amino acids from the C-terminus of the substrate under lower pH conditions (endoprotease activity, neutral pH) [40][41][42][43][44]. Overall, proline at several positions is unfavourable for different cathepsins.…”

Section: Cathepsins Proteases Of the Mhc Class II Presentation Pathwaymentioning

confidence: 99%

Processing and presentation of (pro)‐insulin in the MHC class II pathway: the generation of antigen‐based immunomodulators in the context of type 1 diabetes mellitus

Burster

Boehm

2010

Diabetes Metabolism Res

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SummaryBoth CD4 + and CD8 + T lymphocytes play a crucial role in the autoimmune process leading to T1D. Dendritic cells take up foreign antigens and autoantigens; within their endocytic compartments, proteases degrade exogenous antigens for subsequent presentation to CD4 + T cells via MHC class II molecules. A detailed understanding of autoantigen processing and the identification of autoantigenic T cell epitopes are crucial for the development of antigen-based specific immunomodulators. APL are peptide analogues of auto-immunodominant T cell epitopes that bind to MHC class II molecules and can mediate T cell activation. However, APL can be rapidly degraded by proteases occurring in the extracellular space and inside cells, substantially weakening their efficiency. By contrast, protease-resistant APL function as specific immunomodulators and can be used at low doses to examine the functional plasticity of T cells and to potentially interfere with autoimmune responses. Here, we review the latest achievements in (pro)-insulin processing in the MHC class II pathway and the generation of APL to mitigate autoreactive T cells and to activate Treg cells.

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Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides

Cited by 37 publications

References 31 publications

Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate

Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate

Structural Basis for Inhibition of Cathepsin B Drug Target from the Human Blood Fluke, Schistosoma mansoni

Processing and presentation of (pro)‐insulin in the MHC class II pathway: the generation of antigen‐based immunomodulators in the context of type 1 diabetes mellitus

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