scholarly journals Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8

2000 ◽  
Vol 350 (2) ◽  
pp. 563-568 ◽  
Author(s):  
Henning R. STENNICKE ◽  
Martin RENATUS ◽  
Morten MELDAL ◽  
Guy S. SALVESEN
Keyword(s):  
Peptide Bond ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Peptide Substrates ◽  
Peptide Cleavage ◽  
Fluorescent Peptide ◽  
General Order ◽  
Caspase Family ◽  
General Preference ◽  
Individual Profiles

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P4, P1 and P1´ subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S4 subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S1 and S1´ subsites that flank the scissile peptide bond. The S1 subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P1´ position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P1´ subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

scholarly journals The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B

1989 ◽  
Vol 264 (2) ◽  
pp. 475-481 ◽  
Author(s):  
D Brömme ◽  
A Steinert ◽  
S Friebe ◽  
S Fittkau ◽  
B Wiederanders ◽  
...  
Keyword(s):  
Rat Liver ◽  
Cathepsin L ◽  
Peptide Bond ◽  
Cathepsin S ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Peptide Substrates ◽  
Hydrophobic Residues ◽  
Micromolar Range ◽  
Better Than

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker

2017 ◽  
Vol 95 (6) ◽  
pp. 634-643
Author(s):  
Juliano Alves ◽  
Miguel Garay-Malpartida ◽  
João M. Occhiucci ◽  
José E. Belizário
Keyword(s):  
Amino Acid ◽  
Large Subunit ◽  
Small Subunit ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Caspase 7 ◽  
Caspase Family ◽  
The Impact

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

scholarly journals Action of human liver cathepsin B on the oxidized insulin B chain

1983 ◽  
Vol 213 (2) ◽  
pp. 467-471 ◽  
Author(s):  
M J McKay ◽  
M K Offermann ◽  
A J Barrett ◽  
J S Bond
Keyword(s):  
Amino Acid ◽  
Cathepsin B ◽  
Human Liver ◽  
Degradation Products ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Amino Acid Content ◽  
Peptide Bond ◽  
Cleavage Sites ◽  
Amino Acid Residues

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

scholarly journals Evaluation of the effects of phosphorylation of synthetic peptide substrates on their cleavage by caspase-3 and -7

2021 ◽  
Author(s):  
Izabela Maluch ◽  
Justyna Grzymska ◽  
Scott Snipas ◽  
Guy S Salvesen ◽  
Marcin Drag
Keyword(s):  
Amino Acids ◽  
Caspase 3 ◽  
Amino Acid Residues ◽  
Apoptotic Pathway ◽  
Peptide Substrates ◽  
Peptide Cleavage ◽  
Phosphorylated Proteins ◽  
Execution Phase ◽  
Peptide Length ◽  
The Relationship

Caspases are a family of enzymes that play roles in cell death and inflammation. It has been suggested that in the execution phase of the apoptotic pathway, caspase-3, -6 and -7 are involved. The substrate specificities of two proteases (caspases 3 and 7) are highly similar, which complicates the design of compounds that selectively interact with a single enzyme exclusively. The recognition of residues other than Asp in the P1 position of the substrate by caspase-3/-7 has been reported, promoting interest in the effects of phosphorylation of amino acids in the direct vicinity of the scissile bond. To evaluate conflicting reports on this subject, we synthesized a series of known caspase-3 and -7 substrates and phosphorylated analogs, performed enzyme kinetic assays and mapped the peptide cleavage sites using internally quenched fluorescence peptide substrates. Caspases 3 and 7 will tolerate pSer at the P1 position but only poorly at the P2’ position.  Our investigation demonstrates the importance of peptide length and composition in interpreting sequence/activity relationships. Based on the results, we conclude that the relationship between caspase-3/-7 and their substrates containing phosphorylated amino acids might depend on the steric conditions and not be directly connected with ionic interactions. Thus, the precise effect of phospho-amino acid residues located in the vicinity of the cleaved bond on the regulation of the substrate specificity of caspases remains difficult to predict. Our observations allow to predict that natural phosphorylated proteins may be cleaved by caspases, but only when extended substrate binding site interactions are satisfied.

scholarly journals A systematic series of synthetic chromophoric substrates for aspartic proteinases

1986 ◽  
Vol 237 (3) ◽  
pp. 899-906 ◽  
Author(s):  
B M Dunn ◽  
M Jimenez ◽  
B F Parten ◽  
M J Valler ◽  
C E Rolph ◽  
...  
Keyword(s):  
Structure Function ◽  
Peptide Bond ◽  
Water Soluble ◽  
Animal Origin ◽  
Aspartic Proteinases ◽  
Peptide Substrates ◽  
Representative Selection ◽  
Micro Organisms ◽  
Hydrolysis Of ◽  
Selection Of

The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.

scholarly journals Serum Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) Level as a Potential Biomarker of Cholangiocarcinoma

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 413 ◽  
Author(s):  
Doungdean Tummanatsakun ◽  
Tanakorn Proungvitaya ◽  
Sittiruk Roytrakul ◽  
Temduang Limpaiboon ◽  
Sopit Wongkham ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Transmembrane Helix ◽  
Control Group ◽  
Cleavage Sites ◽  
Dot Blot ◽  
Peptide Cleavage ◽  
Potential Biomarker ◽  
Predicted Signal Peptide ◽  
Healthy Control ◽  
Dot Blot Analysis

Diagnostic and/or prognostic biomarkers for cholangiocarcinoma (CCA) are still insufficient with poor prognosis of patients. To discover a new CCA biomarker, we constructed our secretome database of three CCA cell lines and one control cholangiocyte cell line using GeLC-MS/MS. We selected candidate proteins by five bioinformatics tools for secretome analysis. The inclusion criteria were as follows: having predicted signal peptide or being predicted as non-classically secreted protein; together with having no transmembrane helix and being previously detected in plasma and having the highest number of signal peptide cleavage sites. Eventually, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) was selected for further analysis. To validate APEX1 as a bio-marker for CCA, serum APEX1 levels of 80, 39, and 40 samples collected from CCA, benign biliary diseases (BBD), and healthy control groups, respectively, were measured using dot blot analysis. The results showed that serum APEX1 level in CCA group was significantly higher than that in BBD or healthy control group. Among CCA patients, serum APEX1 level was significantly higher in patients having metastasis than in those without metastasis. The higher level of serum APEX1 was correlated with the shorter survival time of the patients. Serum APEX1 level might be a diagnostic and prognostic biomarker for CCA.

scholarly journals A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid

1991 ◽  
Vol 274 (1) ◽  
pp. 45-48 ◽  
Author(s):  
C G Knight
Keyword(s):  
Amino Acid ◽  
Propionic Acid ◽  
Model Compound ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Fluorescent Substrate ◽  
Peptide Substrates ◽  
Phase Synthesis ◽  
Fluorescent Peptide ◽  
Fluorescent Amino Acid

DL-2-Amino-3-(7-methoxy-4-coumaryl)propionic acid, a new fluorescent amino acid (abbreviated to Amp), has been synthesized to provide an alternative to tryptophan in quenched fluorescent peptide substrates for peptidases. The model compound Ac-DL-Amp-NH2 was intensely fluorescent with an excitation maximum at 328 nm and an emission maximum at 392 nm. Fmoc (fluoren-9-ylmethoxycarbonyl)-DL-Amp was made to allow the solid-phase synthesis of Amp-containing peptides by the Fmoc-polyamide method. The peptide derivative Dnp (2,4-dinitrophenyl)-Pro-Leu-Gly-Pro-DL-Amp-D-Lys was cleaved by thimet peptidase at the Leu-Gly bond, with a 20-fold enhancement of fluorescence. The value of kcat./Km for thimet peptidase was 6.7 x 10(5) M-1.s-1, compared with the value of 2.4 x 10(5) M-1.s-1 for the tryptophan-containing analogue, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys.

scholarly journals Prediction of leader peptide cleavage sites for polypeptides of the thylakoid lumen

1990 ◽  
Vol 18 (11) ◽  
pp. 3417-3417 ◽  
Author(s):  
Christopher J. Howe ◽  
T. Paul Wallace
Keyword(s):  
Leader Peptide ◽  
Cleavage Sites ◽  
Thylakoid Lumen ◽  
Peptide Cleavage

Lachesana tarabaevi, an expert in membrane-active toxins

2016 ◽  
Vol 473 (16) ◽  
pp. 2495-2506 ◽  
Author(s):  
Alexey I. Kuzmenkov ◽  
Maria Y. Sachkova ◽  
Sergey I. Kovalchuk ◽  
Eugene V. Grishin ◽  
Alexander A. Vassilevski
Keyword(s):  
Amino Acid ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Disulfide Bridges ◽  
Venom Protein ◽  
Active Peptides ◽  
Amphiphilic Molecules ◽  
Helical Wheel ◽  
Membrane Active Peptides ◽  
Covalently Linked

In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (∼80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18–79 amino acid residues) comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT) and latartoxins (LtTx). Rpe are short (18 residues) amphiphilic molecules that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b. Isolation of Rpe confirms the validity of the iPQM (inverted processing quadruplet motif) proposed to mark the cleavage sites in spider toxin precursors that are processed into several mature chains. MLys (51 residues) present ‘idealized’ amphiphilicity when modelled in a helical wheel projection with sharply demarcated sectors of hydrophobic, cationic and anionic residues. Four families of CIT (61–79 residues) are the primary weapon of the spider, accounting for its venom toxicity. Toxins from the CIT 1 and 2 families have a modular structure consisting of two shorter Ltc-like peptides. We demonstrate that in CIT 1a, these two parts act in synergy when they are covalently linked. This finding supports the assumption that CIT have evolved through the joining of two shorter membrane-active peptides into one larger molecule.

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