Loosely packed papain prosegment displays inhibitory activity

Gutiérrez‐González, Luis H.; Rojo‐Domínguez, Arturo; Cabrera-González, Nallely E.; Pérez‐Montfort, Ruy; Padilla-Zúñiga, A. Jaqueline

doi:10.1016/j.abb.2005.12.005

Cited by 14 publications

(6 citation statements)

References 30 publications

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“…In general, activation of human cathepsin requires cleavage of its proregion at acidic pH [59]. When cleaved, the proregion exists as a molten globule [60], as we have shown here for crammer under acidic conditions. Nonetheless, the cleaved proregion does not inactivate cathepsin [61].…”

Section: Discussionmentioning

confidence: 54%

A molten globule-to-ordered structure transition of Drosophila melanogaster crammer is required for its ability to inhibit cathepsin

Tseng

Cheng

Chen

et al. 2012

Biochemical Journal

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Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in LTM (long-term memory) formation. The mechanism by which the inhibitory activity is regulated remains unclear. In the present paper we have shown that the oligomeric state of crammer is pH dependent. At neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer, not disulfide-bonded dimer, is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, a condition that is similar to the environment in the lysosome where crammer is probably located. Upon binding to cathepsin L, however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that a cysteine-to-serine point mutation at position 72 (C72S) renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin.

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Section: Discussionmentioning

confidence: 54%

A molten globule-to-ordered structure transition of Drosophila melanogaster crammer is required for its ability to inhibit cathepsin

Tseng

Cheng

Chen

et al. 2012

Biochemical Journal

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“…To date, it appears that the highest enzyme loading in Cu 3 (PO 4 ) 2 complexes is ∼40 wt % papain, an enzyme of the cysteine protease family. Papain from papaya root is negatively charged under the synthesis conditions (pI of 4.4, synthesis at pH 7.4), and the high loadings were achieved with room temperature synthesis and a high concentration of enzyme in the synthesis solution (2 mg mL –1 ). , Similar to our results, 17 wt % HRP was achieved with room temperature synthesis at pH 7.4 over a 3 day period . Room temperature synthesis has also been used to immobilize GOx at 10 wt % .…”

Section: Resultsmentioning

confidence: 99%

Enhancing Enzyme Activity and Immobilization in Nanostructured Inorganic–Enzyme Complexes

et al. 2017

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Understanding the chemical and physical interactions at the interface of protein surfaces and inorganic crystals has important implications in the advancement of immobilized enzyme catalysis. Recently, enzyme-inorganic hybrid complexes have been demonstrated as effective materials for enzyme immobilization. The precipitation of phosphate nanocrystals in the presence of enzymes creates enzyme-Cu(PO)·3HO particles with high surface-to-volume ratios, enhanced activity, and increased stability. Here, we begin to develop a mechanistic understanding of enzyme loading in such complexes. Using a series of enzymes including horseradish peroxidase (HRP), a thermostable alcohol dehydrogenase (AdhD), diaphorase, catalase, glucose oxidase (GOx), and the protein bovine serum albumin (BSA), we identified a correlation between particle synthesis temperature, overall enzyme charge, and enzyme loading. The model enzyme HRP has a high predicted pI of ∼7.5 and maintains an overall positive charge under the synthesis conditions, phosphate buffer pH 7.4. HRP loading in HRP-Cu(PO) complexes was enhanced by 4.2-fold when synthesis was carried out at 37 °C in comparison with synthesis at 4 °C. HRP loading was further enhanced with synthesis at pH 8.0, correlating with a decrease in overall enzyme charge. Proteins with lower predicted pI values and negative overall charge (AdhD, pI of 5.6; diaphorase, pI of 6.8; GOx, pI of 5.2; catalase, pI of 6.9; and, BSA, pI of 5.7) exhibited higher enzyme loadings with 4 °C synthesis, 2.7-, 2.6-, 2.5-, 1.8-, and 1.7-fold protein loading enhancements, respectively. Using HRP as a model system, we also demonstrate that activity increased concomitantly with enzyme loading, and that particle nanostructure was minimally affected by synthesis temperature. Combined, the results presented here demonstrate the control of enzyme loading in enzyme-inorganic particles opening up new possibilities in enzyme and multienzyme catalysis.

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“…The activation of human cathepsin requires the cleavage of its proregion under acidic condition [44]. The cleaved proregion exists as a molten globule [45] with poor inhibitory activity against cathepsin [46]. Unlike the proregion of human cathepsin, the propeptide-like crammer is a strong inhibitor against cathepsin under acidic conditions and the tight binding of crammer is associated with a molten globule-to-ordered structure transition [28].…”

Section: Discussionmentioning

confidence: 99%

Residue-Specific Annotation of Disorder-to-Order Transition and Cathepsin Inhibition of a Propeptide-Like Crammer from D. melanogaster

et al. 2013

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Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB). Far-UV circular dichroism (CD), intrinsic fluorescence, and nuclear magnetic resonance (NMR) spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL) of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins.

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Loosely packed papain prosegment displays inhibitory activity

Cited by 14 publications

References 30 publications

A molten globule-to-ordered structure transition of Drosophila melanogaster crammer is required for its ability to inhibit cathepsin

A molten globule-to-ordered structure transition of Drosophila melanogaster crammer is required for its ability to inhibit cathepsin

Enhancing Enzyme Activity and Immobilization in Nanostructured Inorganic–Enzyme Complexes

Residue-Specific Annotation of Disorder-to-Order Transition and Cathepsin Inhibition of a Propeptide-Like Crammer from D. melanogaster

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