Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin.

Abrahamson, Magnus; Ritonja, Anka; Brown, M A; Grubb, Anders; Machleidt, Werner; Barrett, Alan J.

doi:10.1016/s0021-9258(18)47989-6

Cited by 198 publications

(45 citation statements)

References 28 publications

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“…Motifs involved in the interaction of cystatins with cysteine proteinases are not fully conserved in the cathelin-like domain, however. For example, glycine 9 (chicken cystatin numbering), a conserved residue found in all known sequences of inhibitory cystatins (Abrahamson et al, 1987;Ritonja et al, 1989), is not present in cathelin-like protein. Another highly conserved sequence, QXVXG (residues 53^57), is also not present (Ritonja et al, 1989), indicating that the interaction with cysteine proteinases is di¡erent.…”

Section: Discussionmentioning

confidence: 99%

Antimicrobial and Protease Inhibitory Functions of the Human Cathelicidin (hCAP18/LL-37) Prosequence

Zaiou

Nizet

Gallo³

2003

Journal of Investigative Dermatology

224

180

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Cathelicidins are a class of small cationic peptide antibiotics that are expressed in skin and in other epithelial cells and are an active component of mammalian innate immunity. Human cathelicidin (hCAP18/LL-37) consists of a conserved prosequence called the cathelin-like domain and a C-terminal peptide named LL-37. To date, our understanding of the cathelin-like domain was very limited. To bring insight into the function of this evolutionarily conserved prosequence, we produced recombinant human cathelin-like protein and full-length hCAP18/LL-37 in Escherichia coli. As the cathelin-like protein shares homology with the cystatin family of cysteine protease inhibitors, we first analyzed the effect of the cathelin-like recombinant protein on the cysteine protease cathepsin L. We found that the cathelin-like protein inhibited protease activity. Next, we tested the cathelin-like protein for antimicrobial activity using solid phase radial diffusion and liquid phase killing assays. The cathelin-like prosequence, but not full-length hCAP18/LL-37, killed human pathogens including E. coli and methicillin-resistant Staphylococcus aureus at concentrations ranging from 16 to 32 microM. Together these findings suggest that after proteolytic cleavage the cathelin-like domain can contribute to innate host defense through inhibition of bacterial growth and limitation of cysteine-proteinase-mediated tissue damage. As these dual functions are complementary to the LL-37 peptide released from the C-terminus of full-length hCAP18/LL-37, human cathelicidin represents an elegant multifunctional effector molecule for innate immune defense of the skin.

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Section: Discussionmentioning

confidence: 99%

Antimicrobial and Protease Inhibitory Functions of the Human Cathelicidin (hCAP18/LL-37) Prosequence

Zaiou

Nizet

Gallo³

2003

Journal of Investigative Dermatology

224

180

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“…The principally neuronal phenotype of EPM1 may be a direct and specific consequence of CSTB protein function in neurons. Although the exact role of CSTB has long been studied as a cysteine protease inhibitor, its precise physiological function(s) remains unclear (21). Alternatively, it is possible that the CSTB protein has the same role in all cells but that there is a lower threshold for its absence in neurons.…”

Section: Discussionmentioning

confidence: 99%

Altered Spacing of Promoter Elements Due to the Dodecamer Repeat Expansion Contributes to Reduced Expression of the Cystatin B Gene in EPM1

Lalioti

Scott

Antonarakis

1999

Human Molecular Genetics

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Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1; MIM 254800) is an autosomal recessive disorder characterized by seizures, myoclonus and progression to cerebellar ataxia. EPM1 arises due to mutations in the cystatin B (CSTB) gene which encodes a cysteine proteinase inhibitor. Only a minority of EPM1 alleles carry point mutations, while the majority contain large expansions of the dodecamer CCCCGCCCCGCG repeat which is present at two to three copies in normal individuals. The dodecamer repeat is located in the 5' flanking region of the CSTB gene, presumably in its promoter. The pathological repeat expansion results in a reduction in CSTB mRNA, which may be cell specific. To elucidate the mechanism of this reduction of gene expression, we have studied the putative CSTB promoter in vitro. A 3.8 kb fragment, containing the putative promoter with a 600 bp repeat expansion, showed a 2- to 4-fold reduction in luciferase activity compared with an identical fragment with a normal repeat; this reduction was observed only in certain cell types. Introduction of heterologous DNA fragments of 730 and 1000 bp into the normal promoter, instead of the repeat expansion, showed similarly reduced activity. Terminal deletions of the promoter implicate a putative AP-1 binding site, upstream of the repeat, in CSTB transcription activation. We propose that a novel mechanism of pathogenesis, the altering of the spacing of transcription factor binding sites from each other and/or the transcription initiation site due to repeat expansion, is among the causes of reduction in CSTB expression and thus EPM1.

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“…It has previously been shown that the N-terminal 11 amino acid residues of cystatin C are important for its high-affinity binding to papain, a plant cysteine proteinase, and it has been suggested that the polypeptide chain region around the Gly-11 residue might serve as a substrate-like binding region for cysteine proteinases (Abrahamson et al, 1987b). Peptides with amino acid sequences identical with that of the N-terminal segment of cystatin C are indeed good substrates for papain, being cleaved at the bond following residue Gly-l after incubation with the enzyme (Abrahamson et al, 1987b;Grubb et al, 1990). A peptidyldiazomethane mimicking part of the proposed substratelike-binding region of cystatin C displays irreversible inhibition of papain and a streptococcal cysteine proteinase and appears to have a potential as an antimicrobial drug (Bjorck et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

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

Abrahamson

Mason

Hansson

et al. 1991

Biochemical Journal

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166

139

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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.

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Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin.

Cited by 198 publications

References 28 publications

Antimicrobial and Protease Inhibitory Functions of the Human Cathelicidin (hCAP18/LL-37) Prosequence

Antimicrobial and Protease Inhibitory Functions of the Human Cathelicidin (hCAP18/LL-37) Prosequence

Altered Spacing of Promoter Elements Due to the Dodecamer Repeat Expansion Contributes to Reduced Expression of the Cystatin B Gene in EPM1

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

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