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

1998 ◽  
Vol 330 (2) ◽  
pp. 833-838 ◽  
Author(s):  
W. Robert MASON ◽  
Katia SOL-CHURCH ◽  
Magnus ABRAHAMSON
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Cathepsin L ◽  
Specific Inhibitor ◽  
Terminal Segment ◽  
Cathepsin S ◽  
Amino Acid Substitutions ◽  
Cysteine Proteinases ◽  
Aromatic Residues ◽  
Human Cystatin C

We used site-directed mutagenesis to alter the specificity of human cystatin C, an inhibitor with a broad reactivity against cysteine proteinases. Nine cystatin C variants containing amino acid substitutions in the N-terminal (L9W, V10W, V10F and V10R) and/or the C-terminal (W106G) enzyme-binding regions were designed and produced in Escherichia coli. It was discovered that the inhibition profile of the cystatin could be altered by changing residues 9 and 10, which are proposed to bind in the S3 and S2 substrate-binding pockets respectively of the enzymes. All of the variants with substitutions in the N-terminal segment displayed decreased binding to cathepsins B and H, indicating that the S3 and S2 pockets of these enzymes cannot easily accommodate large aromatic residues. The introduction of a charged residue into S2 (variant V10R) created a more specific inhibitor to distinguish cathepsin B from cathepsin H. Cathepsin L showed a preference for larger aromatic residues in S2. In contrast, cathepsin S preferred phenylalanine to valine in S2, but bound less tightly to the V10W cystatin variant. The latter variant proved to be valuable for discriminating between cathepsin L and cathepsin S (Ki 2.4 and 190 pM respectively). The equilibrium dissociation constant of the complex between cathepsin L and variant L9W/W106G showed little difference in affinity from that of the cathepsin L complex with the singly substituted W106G variant. In contrast, the L9W/W106G variant displayed increased specificity for cathepsin S with a Ki of 10 pM. Our results clearly indicate differences in the specificity of interaction between the N-terminal region of cystatin C and cathepsins B, H, L and S, and that, although cystatin C has evolved to be a good inhibitor of all of the mammalian cysteine proteinases, more specific inhibitors of the individual enzymes can be engineered.

scholarly journals Human cystatin C. role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase

1991 ◽  
Vol 273 (3) ◽  
pp. 621-626 ◽  
Author(s):  
M Abrahamson ◽  
R W Mason ◽  
H Hansson ◽  
D J Buttle ◽  
A Grubb ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Cysteine Proteinase ◽  
Terminal Segment ◽  
Cysteine Proteinase Inhibitor ◽  
Cathepsin H ◽  
Leucocyte Elastase ◽  
Human Cystatin C ◽  
Binding Pockets ◽  
Human Cystatin

Leucocyte elastase in catalytic amounts was observed to rapidly cleave the Val-10-Gly-11 bond of the human cysteine-proteinase inhibitor cystatin C at neutral pH. The resulting modified inhibitor had size and amino acid composition consistent with a cystatin C molecule devoid of the N-terminal Ser-1-Val-10 decapeptide. Leucocyte-elastase-modified cystatin C had more than 240-fold lower affinity than native cystatin C for papain. Removal of the N-terminal decapeptide of human cystatin C also decreased inhibition of human cathepsins B and L by three orders of magnitude, but decreased inhibition of cathepsin H by only 5-fold. A tripeptidyldiazomethane analogue of of the N-terminal portion of cystatin C was a good inhibitor of cathepsins B and L but a poor inhibitor of cathepsin H. It therefore appears that amino acid side chains of the N-terminal segment of cystatin C bind in the substrate-binding pockets of cathepsins B and L but not in those of cathepsin H. It is argued that the N-terminal cystatin C interaction with cathepsin B is physiologically important and hence that leucocyte elastase could have a function as a regulator of extracellular cysteine-proteinase inhibitory activity at sites of inflammation.

scholarly journals Differential Regulation of Cathepsin S and Cathepsin L in Interferon γ–treated Macrophages

2003 ◽  
Vol 197 (2) ◽  
pp. 169-179 ◽  
Author(s):  
Courtney Beers ◽  
Karen Honey ◽  
Susan Fink ◽  
Katherine Forbush ◽  
Alexander Rudensky
Keyword(s):  
Cathepsin L ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Interferon Γ ◽  
Cathepsin S ◽  
Antigen Presenting Cell ◽  
Relative Contribution ◽  
Helper Cell Type ◽  
Ifn Γ ◽  
Antigen Presenting

Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mϕs) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-γ–stimulated Mϕs. In addition, our studies show that the level of catL activity is significantly decreased in Mϕs cultured in the presence of IFN-γ whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-γ–stimulated peritoneal Mϕs. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mϕs exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-γ. Thus, during a T helper cell type 1 immune response catL inhibition in Mϕs results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.

scholarly journals Quantification of cathepsins B and L in cells

1998 ◽  
Vol 332 (2) ◽  
pp. 499-505 ◽  
Author(s):  
Ruye XING ◽  
Adele K. ADDINGTON ◽  
Robert W. MASON
Keyword(s):  
Active Site ◽  
Total Protein ◽  
Cathepsin B ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cathepsin L ◽  
Breast Tumour ◽  
Cathepsin S ◽  
Cysteine Proteinases ◽  
Breast Tumour Cells

A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diazomethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.

The role of the Val57 amino-acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2-L1-beta3 segments of wild-type human cystatin C and its mutants

Biopolymers ◽  
2009 ◽  
Vol 91 (5) ◽  
pp. 373-383 ◽  
Author(s):  
Sylwia Rodziewicz-Motowidło ◽  
Justyna Iwaszkiewicz ◽  
Renata Sosnowska ◽  
Paulina Czaplewska ◽  
Emil Sobolewski ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Amino Acid Residue ◽  
Wild Type ◽  
Conformational Studies ◽  
Human Cystatin C ◽  
Human Cystatin

Interaction of serum amyloid A with human cystatin C-assessment of amino acid residues crucial for hCC-SAA formation (part II)

2013 ◽  
Vol 26 (9) ◽  
pp. 415-425 ◽  
Author(s):  
Marta Spodzieja ◽  
Monika Rafalik ◽  
Aneta Szymańska ◽  
Aleksandra S. Kołodziejczyk ◽  
Paulina Czaplewska
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Serum Amyloid A ◽  
Serum Amyloid ◽  
Amino Acid Residues ◽  
Amyloid A ◽  
Human Cystatin C ◽  
Human Cystatin

scholarly journals Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi

1996 ◽  
Vol 318 (2) ◽  
pp. 395-399 ◽  
Author(s):  
Gilles LALMANACH ◽  
Roger MAYER ◽  
Carole SERVEAU ◽  
Julio SCHARFSTEIN ◽  
Francis GAUTHIER
Keyword(s):  
Trypanosoma Cruzi ◽  
Cathepsin B ◽  
Active Sites ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Irreversible Inhibitors ◽  
Human Cystatin C ◽  
Spacer Arm ◽  
Biotin Labelling

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin–peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.

scholarly journals Human low-Mr kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases

1986 ◽  
Vol 234 (2) ◽  
pp. 429-434 ◽  
Author(s):  
G Salvesen ◽  
C Parkes ◽  
M Abrahamson ◽  
A Grubb ◽  
A J Barrett
Keyword(s):  
Amino Acid ◽  
Inhibitory Activity ◽  
Cathepsin L ◽  
Limited Proteolysis ◽  
Cysteine Proteinases ◽  
Disulphide Bonds ◽  
N Terminus ◽  
Domain 3 ◽  
The Individual ◽  
Activity Domain

We point out that human low-Mr kininogen contains three cystatin-like sequences, rather than two, as had previously been thought. The protein was purified by affinity chromatography on carboxymethyl-papain-Sepharose, and subjected to limited proteolysis by trypsin and chymotrypsin. Fragments were isolated, and three corresponding to the individual cystatin-like domains were identified. By comparison with the known amino acid sequence of the protein they were numbered 1 to 3 from the N-terminus. Domain 1 was not found to have any inhibitory activity for cysteine proteinases, which is consistent with the absence of residues that are highly conserved in inhibitors of the cystatin superfamily, and have previously been suggested to be essential for activity. Domain 2 was a good inhibitor of chicken calpain, and also papain and cathepsin L. Domain 3 showed negligible inhibition of calpain, but inhibited papain and cathepsin L strongly. The probable arrangement of disulphide bonds in the heavy chain of low-Mr kininogen is deduced from the homology with the cystatins and other evidence contained in the present paper.

scholarly journals Charting the sequence-activity landscape of peptide inhibitors of translation termination

2021 ◽  
Vol 118 (10) ◽  
pp. e2026465118
Author(s):  
Chetana Baliga ◽  
Tyler J. Brown ◽  
Tanja Florin ◽  
Sarah Colon ◽  
Vallari Shah ◽  
...  
Keyword(s):  
Amino Acid ◽  
Translation Termination ◽  
Expression System ◽  
Antibacterial Properties ◽  
Specific Inhibitor ◽  
Antibacterial Peptide ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Release Factors ◽  
Exit Tunnel

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api’s activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

scholarly journals Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition

1993 ◽  
Vol 291 (1) ◽  
pp. 123-129 ◽  
Author(s):  
A Hall ◽  
H Dalbøge ◽  
A Grubb ◽  
M Abrahamson
Keyword(s):  
Cystatin C ◽  
Equilibrium Constants ◽  
Terminal Segment ◽  
Site Directed Mutagenesis ◽  
Side Chains ◽  
Wild Type ◽  
Human Cystatin C ◽  
Evolutionarily Conserved ◽  
Cysteine Endopeptidases ◽  
Human Cystatin

Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (Ki) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed Ki values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11. Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the Ki values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The Ki values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment.

scholarly journals Adaptation of Reovirus to Growth in the Presence of Protease Inhibitor E64 Segregates with a Mutation in the Carboxy Terminus of Viral Outer-Capsid Protein ς3

2001 ◽  
Vol 75 (7) ◽  
pp. 3197-3206 ◽  
Author(s):  
Daniel H. Ebert ◽  
J. Denise Wetzel ◽  
David E. Brumbaugh ◽  
Stacey R. Chance ◽  
Laura E. Stobie ◽  
...  
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Cathepsin L ◽  
Specific Inhibitor ◽  
Serial Passage ◽  
Outer Capsid Protein ◽  
Strain Type ◽  
Reassortant Viruses ◽  
Type 3 ◽  
Outer Capsid

ABSTRACT Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of ς3 protein and proteolytic cleavage of μ1/μ1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 μM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein ς3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of ς3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of ς3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in ς3 protein containing amino acid 354 influences susceptibility of ς3 to proteolysis during reovirus disassembly.

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