3,4‐Dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase <i>b</i>

Rusbridge, Nicholas M.; Beynon, Robert J.

doi:10.1016/0014-5793(90)80991-q

Cited by 17 publications

(18 citation statements)

References 5 publications

(4 reference statements)

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“…It is known that these inhibitors block protease activity by direct alkylation or acylation of the protease active site (24)(25)(26). Moreover, TPCK, TLCK, and DCIC alter the activity of proteins in addition to proteases such as glycogen phosphorylase b, TFIIIC, and Ets-1 through direct modifications (27)(28)(29). Furthermore, NF-KB DNA binding activity is redoxsensitive and can be inhibited by alkylation within its DNA binding domain (30,31).…”

Section: Discussionmentioning

confidence: 99%

Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.

Finco

Beg

Baldwin³

1994

Proc. Natl. Acad. Sci. U.S.A.

274

210

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The ubiquitous transcription factor NF-KB is regulated by its cytoplasmic inhibitor IKB. A variety of cellular stimuli cause the dissociation of NF-#cB from IKB, allowing NF-KB to translocate to the nucleus and regulate gene expression. Although the activation ofNF-KB in vivo is associated with the phosphorylation and degradation of IKBa, it has remained unclear how each of these events contributes to this process. Recently, studies utilizing protease inhibitors have suggested that the proteolysis of IKBa is a necessary event in the activation of NF-ucB. We demonstrate in this study that these and an additional protease inhibitor also completely repress inducible phosphorylation of IKBa. This surprising result suggests a more complex role of proteases in NF-KB activation. In addition, data presented here indicate that many of these inhibitors also directly modify NF-KB and inhibit its DNA binding activity. Due to the pleiotropic effects of these protease inhibitors, it is difficult to conclude from their use how IKBa phosphorylation and degradation contribute to NF-KB activation. In the present study, a more direct approach demonstrates that phosphorylation of IKBa alone is not sufficient for NF-KB activation.

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Section: Discussionmentioning

confidence: 99%

Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.

Finco

Beg

Baldwin³

1994

Proc. Natl. Acad. Sci. U.S.A.

274

210

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“…Providing it is used at reasonable concentrations, 3,4-dichloroisocoumarin acts as a mechanism-based inhibitor of serine proteases Rusbridge and Beynon, 1990) and crystallographic data have shown it to bind covalently to the catalytic serine and/or histidinc residue (Vijayalakshmi et al, 1991). It is an effective inhibitor of some MCP (Orlowski and Michaud, 1989;Mason, 1990).…”

Section: Discussionmentioning

confidence: 99%

“…1) except at very high 3,4-dichloroisocoumarin concentrations, at which this compound becomes a general alkylating reagent (Rusbridge and Beynon, 1990). Activity with Boc-Leu-Ser-Thr-Arg-NH-Mec was stimulated by 3,4-dichloroisocoumarin then decreased with time, but it was not possible to inactivate the Boc-Leu-SerThr-Arg-NH-Mec activity with 3,4-dichloroisocoumarin.…”

Section: Inhibition By 34-dichloroisocoumarinmentioning

confidence: 99%

Use of serine‐protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex

Djaballah

Harness

Savory

et al. 1992

European Journal of Biochemistry

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The multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy-group side of basic, hydrophobic or acidic amino acid residues. Orginally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for socalled trypsin-like activity with Boc-Leu-Ser-Thr-Arg-NH-Mec (Mec, 4-methylcoumarin; Boc, tbutoxycarbonyl), for chymotrypsin-like activity with Ala-Ala-Phe-NH-Mec and Suc-Leu-Leu-ValTyr-NH-MEc (SUC, succinyl), and peptidyl-glutamylpeptide hydrolase activity with Cbz-Leu-LeuGlu-Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin-like activity and two for each of the others. The activities were tested with a variety of serine-protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4-(2-aminoethy1)benzenesulphonyl fluoride, which selectivity inactivates the trypsin-like activity, and 3,4-dichloroisocoumarin which inhibits chymotrypsin-like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133 -41411 of peptidylglutamylpeptide hydrolase activity. Thc three activities inhibited by 3,4-dichloroisocoumarin can easily be distinguished by the effects of chyrnostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.

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“…Recently, it has been shown that 3,4-dichloroisocoumarin also inactivates non-proteolytic enzymes, by modification of multiple residue(s) within the enzyme protein, e.g. glycogen phosphorylase (Rusbridge and Beynon, 1990). Similarly, in the TherrnopIasma proteasome, more than one amino acid residue seems to be modified by 3,4-dichloroisocoumarin, since the isoelectric points of both, a and subunits are changed after reaction with the inhibitor, and furthermore, the subunits are detectable at different p I after isoelectric focusing.…”

Section: Discuss I 0 Nmentioning

confidence: 99%

Biochemical properties of the proteasome from Thermoplasma acidophilum

Dahlmann¹,

Kuehn²,

Grziwa³

et al. 1992

European Journal of Biochemistry

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We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90°C. The enzyme activity is enhanced severalford by Mg2+ and Ca2+ at 25–500 mM. This increase in activity results primarily from a change in Km. The serine‐proteinase inhibitors diisopropylfluorophosphate and 3,4‐dichlorosiocoumarin irreversibly inhibit the enzyme, obviously by modification of both the α and β subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz‐Leu‐Leu‐CH2Cl and Cbz‐Ala‐Ala‐Phe‐Ch2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.

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3,4‐Dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b

Cited by 17 publications

References 5 publications

Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.

Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.

Use of serine‐protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex

Biochemical properties of the proteasome from Thermoplasma acidophilum

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