Inhibition of Mammalian Legumain by Some Cystatins Is Due to a Novel Second Reactive Site

Alvarez-Fernandez, Marcia; Barrett, Alan J.; Gerhartz, Bernd; Dando, Pam M.; Ni, Jian; Abrahamson, Magnus

doi:10.1074/jbc.274.27.19195

Cited by 257 publications

(234 citation statements)

References 52 publications

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“…Previous studies showed that some typical cysteine proteinase inhibitors, as, for example, cystatins and E64 (44,45), cannot block the proteolytic activity of IdeS (6). Comparison of the crystal structures of IdeS and other proteinases reveals that overall differences between the active-site cleft of IdeS and other papain-like cysteine proteinases are responsible for the inability to affect IdeS activity.…”

Section: Discussionmentioning

confidence: 99%

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG

Wenig

Chatwell²,

Pawel‐Rammingen³

et al. 2004

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

133

109

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Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-Å resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.Streptococcus pyogenes ͉ Mac-1

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Section: Discussionmentioning

confidence: 99%

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG

Wenig

Chatwell²,

Pawel‐Rammingen³

et al. 2004

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

133

109

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“…Inspection of the aligned sequences of these isoforms in their surface regions focused attention on the ␤5-␤6 loop, positioned in the lower crown region (residues 71-76 containing the sequence Ile-AspAsn-Ser-Ile). This part of the sequence is similar to the consensus sequence (S/T)N(D/S)(M/I) found in three inhibitory cystatins C, E, and F ( Table 4) that bind to AEP in the nanomolar range (40). Interestingly, in macrocypin 4, the residue at position 72 is Lys, in contrast to the equipositioned Asn in macrocypins 1 and 3.…”

Section: Tablementioning

confidence: 80%

“…Thus, Asn-72 in macrocypins and Asn-69 in clitocypins are confirmed to be the residues responsible for the inhibition of AEP. Mycocypins are, in this respect, similar to cystatin C, which has two different binding sites, one for papain-like proteases and another for AEP (40).…”

Section: Table 3 Inhibition Constants Of Cathepsin V By Macrocypin Cmentioning

confidence: 99%

“…Interestingly, in macrocypin 4, the residue at position 72 is Lys, in contrast to the equipositioned Asn in macrocypins 1 and 3. To verify the role of the residues in these regions, like Alvarez-Fernandez et al (40) in the case of cystatin C, we introduced mutations in the inhibitory sequence. The residues that differ between macrocypins 1 and 4 in the ␤5-␤6 loop were Clitocypin loops are shown as sticks.…”

Section: Tablementioning

confidence: 99%

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Versatile Loops in Mycocypins Inhibit Three Protease Families

Renko

Sabotič

Mihelič

et al. 2010

Journal of Biological Chemistry

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Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue. The binding is associated with a peptide-bond flip of glycine that occurs before or concurrently with the inhibitor docking. Mycocypins possess a ␤-trefoil fold, the hallmark of Kunitz-type inhibitors. It is a tree-like structure with two loops in the root region, a stem comprising a six-stranded ␤-barrel, and two layers of loops (6 ؉ 3) in the crown region. The two loops that bind to cysteine cathepsins belong to the lower layer of the crown loops, whereas a single loop from the crown region can inhibit trypsin or asparaginyl endopeptidase, as demonstrated by site-directed mutagenesis. These loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.Inhibition of foreign protease activity is a widespread defense mechanism in plants against their pests, pathogens, and parasites (1). Protein inhibitors of proteases are present in a variety of plant tissues. They can be deployed alone or together with a variety of small molecules (2). It has been known for a long time that the expression of protease inhibitors is increased in injured plant leaves (3) and that their expression can be induced as a response to attack by insects or pathogens (2).Given the negative environmental effects of chemical pesticides used in crop protection, it is important to explore alternative approaches, such as the incorporation of genes encoding protease inhibitors into plants. Transgenic plants expressing various protease inhibitors have shown enhanced levels of insect resistance; however, the adaptive capacity of insect digestive proteases limits the use of single protease inhibitors (4, 5). The use of hybrid protease inhibitors with multiple inhibitory activity could, however, affect the functional properties of the fused inhibitors (6). Incorporation of genes encoding a range of protease inhibitors is to run the risk of deleterious modification of plants, but the use of a single protease inhibitor with versatile functionality could be the way forward.With these in mind, we have undertaken structural and mechanistic studies of the cysteine protease inhibitors clitocypin (Clt) 3 from mushroom Basidiomycetes Clitocybe nebularis and macrocypins (Mcp) from Macrolepiota procera. Based on the lack of sequence similarity to other protease inhibitors, they form separate protease inhibitor famil...

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“…Na literatura. temos que legumaína é uma endopeptidase caracterizada por não ser afetada por esse clássico inibidor, porém por outras cistatinas (Alavarez-Fernandez et al, 1999). A isoforma da Fração Solúvel caracterizada nesse trabalho possivelmente pertence a essa família de enzimas, porém testes com outras cistatinas deverão ser realizados para se confirmar que as cisteíno proteases da Fração Solúvel são cisteíno proteases legumaína-like.…”

Section: Methodsunclassified

Caracterização enzimática de isoformas de cisteíno protease de Anticarsia gemmatalis (Hübner, 1818)

et al. 2011

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RESUMOIsoformas de cisteíno protease obtidas do intestino médio de lagartas de 5º instar de Anticarsia gemmatalis (Hübner, 1818) foram caracterizadas. A isoforma solúvel foi chamada de Fração Solúvel enquanto a isoforma ligada à membrana celular, de Fração Insolúvel. As maiores atividades foram observadas em pH 3,6 a 45° C para a Fração Solúvel e pH 4,6 a 50° C para a Fração Insolúvel. Ao analisar o efeito de modificadores químicos, a Fração Solúvel mostrou-se insensível à aprotinina e E-64, porém teve sua atividade aumentada pela adição de EDTA e levemente inibida pela adição de íons Ca 2+ , mostrando se tratar de enzimas independentes de íons metálicos para sua atividade. A Fração Insolúvel também se mostrou insensível à aprotinina, porém teve sua atividade parcialmente inibida por E-64. A adição de EDTA levou a uma redução nos valores de atividade, demonstrando a necessidade de íons metálicos para a atividade dessas enzimas, porém não se trata de enzimas cálcio-dependentes, uma vez que sua atividade foi reduzida com a adição desse íon. Os valores de K M app e V máx app foram, respectivamente, 0,6398 mM e 42,556 nM s -1 para Fração Solúvel e 0,0413 mM e 10,854 nM s -1 para Fração Insolúvel. Esses resultados fornecem evidências da presença de cisteíno protease solúvel e ligada à membrana celular do intestino de lagartas de A. gemmatalis. O conhecimento e a caracterização das principais classes de proteases presentes no trato digestivo da lagarta da soja, bem como a interação dessas enzimas com inibidores de protease têm uma importante consequência aos programas de melhoramento de soja.Termos para indexação: Proteases digestivas, Lepidoptera, controle de pragas, inibição de proteases. ABSTRACTCysteine protease isoforms obtained from the midgut of 5 th instar Anticarsia gemmatalis (Hübner, 1818) larvae were characterized. The soluble enzyme isoform was called Soluble Fraction and cell membrane-bound enzyme isoform, the Insoluble Fraction. The highest activities were observed at pH 3.6 and 45° C for the Soluble Fraction and pH 4.6 and 50° C for the Insoluble Fraction. Analyzing the effect of chemical modifiers, the Soluble Fraction was shown to be insensitive to aprotinin and E-64, although its activity was increased by the addition of EDTA and slightly inhibited by the addition of Ca 2+ , showing to be enzymes independent of metal ions to its activity. The Insoluble Fraction was also insensitive to aprotinin, but its activity was partially inhibited by E-64. The addition of EDTA reduced the activity values, demonstrating the need for metal ions for the activity of these enzymes, but they are not calcium-dependent enzymes, since their activity was reduced with the addition of this ion. The values of K M app and V max app were, respectively, 0.6398 mM and 42.556 nM s -1 for Soluble Fraction and 0.0413 mM and 10.854 nM s -1 for Insoluble Fraction. These results provide evidence for the presence of soluble and cell membrane-bound cysteine proteases in the gut of A. gemmatalis larvae. The knowledge and characterizatio...

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Inhibition of Mammalian Legumain by Some Cystatins Is Due to a Novel Second Reactive Site

Cited by 257 publications

References 52 publications

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG

Versatile Loops in Mycocypins Inhibit Three Protease Families

Caracterização enzimática de isoformas de cisteíno protease de Anticarsia gemmatalis (Hübner, 1818)

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