Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases

Mason, W. Robert; Sol‐Church, Katia; Abrahamson, Magnus

doi:10.1042/bj3300833

Cited by 42 publications

(37 citation statements)

References 19 publications

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“…The Binding Site for Papain-like Peptidases-The papainbinding site in cystatins is constituted by three well-conserved segments: the flexible N-terminal part, a hairpin loop in the central region (L1), and another toward the C-terminal end (L2) of the sequence (33)(34)(35)(36)(37)(38)(39)(40)(41)(42). These segments form a tripartite wedge-shaped edge (7,8,10,11,43) that enters the catalytic site in a substrate-like manner (10,44).…”

Section: Crystallization Of Cystatin D and Crystal Data Collection-mentioning

confidence: 99%

Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

Alvarez-Fernandez

Liang

Abrahamson

et al. 2005

Journal of Biological Chemistry

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Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg 26 -cystatin D and solved its structures at room temperature and at cryo conditions to 2.5-and 1.8-Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded antiparallel ␤-sheet wrapped around a five-turn ␣-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumainrelated enzymes.

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Section: Crystallization Of Cystatin D and Crystal Data Collection-mentioning

confidence: 99%

Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

Alvarez-Fernandez

Liang

Abrahamson

et al. 2005

Journal of Biological Chemistry

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“…The activity of the modified cystatin C is reduced, but not uniformly: The affinity for cathepsins B and L is more than three orders of magnitude lower, while for cathepsin H it is only five-fold lower (although this appears to underestimate the importance of this region for this enzyme [Hall et al, 1995]). Therefore, the N-terminal region of cystatin C likely binds in the substrate-binding pockets of cathepsins B and L, but makes little contribution to cystatin C binding to cathepsin H. To examine the contributions of the individual N-terminal side-chains to binding, investigators have used site-directed mutagenesis to replace residues 8-10 with glycine or other amino acids, either singly or in combination (Hall et al, 1995;Mason et al, 1998). Residue 10 was found to be responsible for the main contribution to binding affinity for cathepsins B, H, L, and S. Most V10 substitutions tested caused a decrease in the Ki.…”

Section: (I) N-terminal Domainmentioning

confidence: 99%

Salivary (SD-Type)Cystatins:OverOneBillionYears in theMaking—But toWhatPurpose?

Dickinson

2002

Critical Reviews in Oral Biology & Medicine

103

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The cystatin superfamily is comprised of a large group of ancestrally related proteins, most of which are potent inhibitors of cysteine peptidases (CPs). Human saliva contains relatively abundant proteins that are related ancestrally in sequence to the cystatin superfamily. These proteins are now commonly referred to as 'salivary cystatins', although this is something of a misnomer (see below). It has been several years since the publication of the last comprehensive reviews that focused on the salivary cystatins (Bobek and Levine, 1992;Henskens et al., 1996c). Since then, significant advances have been made regarding the gene organization, protein structure, and properties of human and rat proteins. Therefore, a major purpose of this article is to review this recent work. What is the function of the 'salivary cystatins'? Despite a great deal of research effort, we still do not have a definitive answer to this question. A major impediment is the lack of a disease to associate with a specific defect in a gene: Functionality must be inferred from circumstantial evidence. A central premise of this review is that clues to 'salivary cystatin' function are contained within their phylogenetic history, and that by looking at established or likely functions of their 'siblings' and 'cousins', one can assess the likelihood of this function(s) continuing into the 'salivary cystatins'. A comprehensive review of the large cystatin superfamily is beyond the scope of a single article. This review will aim to outline the main branches of the superfamily and the common elements of their structure and function, but will pay closest attention to the nearest relatives of the 'salivary cystatins'.(1) BACKGROUND Scission of peptide bonds is an essential reaction in living cells, and there is a considerable number of peptidases that catalyze this reaction with various degrees of specificity. CPs use a cysteine residue in the active site as the nucleophile in the reaction (see Dickinson, 2002, and references therein). Release of proteolytic enzymes outside of their normal compartment has the potential to cause serious degradation and pathology-an effect taken advantage of by pathogenic organisms from a wide range of phyla. Therefore, control of proteolytic enzyme activity is mandatory. Building upon a very early report (see Barrett et al., 1986, for a review of this earlier literature) of a bovine trypsin inhibitor in chicken egg white, a search for egg white inhibitors of plant proteinases (the CPs ficin and papain) identified a relatively small (12.7 kDa) protein that was a potent inhibitor (Ki = 10 nM). The term cystatin was coined for this protein, which was shown also to inhibit mammalian CPs of the papain superfamily, including cathepsins B, C, H, and L. Cystatins form 1:1 reversible complexes with CPs, in competition with the substrate, but in some cases the binding is so tight as to be physiologically irreversible. (2) THE CYSTATIN SUPERFAMILYExamination of mammalian tissues and serum revealed several CP inhibitors ranging in si...

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“…Several approaches based on structure/function models have been used over the years to improve the inhibitory potency of protease inhibitors against specific proteases, including sitedirected mutagenesis of specific amino acids or amino acid strings (e.g. Qasim et al, 1997;Mason et al, 1998;Ogawa et al, 2002;Pavlova and Bjö rk, 2003) and molecular phage display procedures involving random mutagenesis in specific regions of the inhibitor sequence (e.g. Laboissiere et al, 2002;Ceci et al, 2003;Melo et al, 2003;Stoop and Craik, 2003).…”

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confidence: 99%

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

et al. 2008

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Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatininsensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.

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Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases

Cited by 42 publications

References 19 publications

Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

Salivary (SD-Type)Cystatins:OverOneBillionYears in theMaking—But toWhatPurpose?

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

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