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

ABSTRACT: 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 P(4), P(1) and P(1)' 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 S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) 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 P(1)' 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 P(1)' 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.

SUBMITTER: Stennicke HR

PROVIDER: S-EPMC1221285 | biostudies-other | 2000 Sep

REPOSITORIES: biostudies-other

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Project description:Protease biology is intimately linked to the functional consequences of substrate cleavage events. Human caspases are a family of 12 fate-determining cysteine proteases that are best known for driving cell death, either apoptosis or pyroptosis. More recently, caspases have been shown to be involved in other cellular remodeling events as well including stem cell fate determination, spermatogenesis, and erythroid differentiation. Recent global proteomics methods enable characterization of the substrates that caspases cleave in live cells and cell extracts. The number of substrate targets identified for individual caspases can vary widely ranging from only a (few) dozen targets for caspases-4, -5, -9, and -14 to hundreds of targets for caspases-1, -2, -3, -6, -7, and -8. Proteomic studies characterizing the rates of target cleavage show that each caspase has a preferred substrate cohort that sometimes overlaps between caspases, but whose rates of cleavage vary over 500-fold within each group. Determining the functional consequences of discrete proteolytic events within the global substrate pool is a major challenge for the field. From the handful of individual targets that have been studied in detail, there are only a few so far that whose single cleavage event is capable of sparking apoptosis alone, such as cleavage of caspase-3/-7 and BIMEL, or for pyroptosis, gasdermin D. For the most part, it appears that cleavage events function cooperatively in the cell death process to generate a proteolytic synthetic lethal outcome. In contrast to apoptosis, far less is known about caspase biology in non-apoptotic cellular processes, such as cellular remodeling, including which caspases are activated, the mechanisms of their activation and deactivation, and the key substrate targets. Here we survey the progress made in global identification of caspase substrates using proteomics and the exciting new avenues these studies have opened for understanding the molecular logic of substrate cleavage in apoptotic and non-apoptotic processes.

Project description:The application of D-stereospecific proteases (DSPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable DSPs covering multiple substrate specificities. To identify DSPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl-D-arginyl-D-alanyl-p-nitroanilide was developed. By monitoring both UV/vis-absorbance and fluorescence signals at the same time it allows to detect two distinct D-amino acid substrate specificities simultaneously and separately with respect to the individual specificities. In order to identify novel DSP specificities for synthesis applications, DSPs specific for D-arginine were of special interest due to their potential ability as catalysts for substrate mimetics-mediated peptide and protein ligations. D-alanine in the substrate served as positive control and reference based on its known acceptance by numerous DSPs. In silico analysis suggested that DSPs are predominantly present in gram-positive microorganisms, therefore this study focused on the bacilli strains Bacillus thuringiensis and Bacillus subtilis as potential hosts of D-Arg-specific DSPs. While protease activities toward D-alanine were found in both organisms, a novel and so far unknown D-arginine specific DSP was detected within the culture supernatant of B. thuringiensis. Enrichment of this activity via cation exchange and size exclusion chromatography allowed isolation and further characterization of this novel enzyme consisting of a molecular mass of 37.7 kDa and an enzymatic activity of 8.3 U mg-1 for cleaving the D-Arg|D-Ala bond in the detecting substrate. Independent experiments also showed that the identified enzyme shows similarities to the class of penicillin binding proteins. In future applications this enzyme will be a promising starting point for the development of novel strategies for the semisynthesis of all-L-proteins.

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

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