Modelling family 2 cystatins and their interaction with papain

Nandy, Suman Kumar; Bhuyan, Rajabrata; Seal, Alpana

doi:10.1080/07391102.2012.706403

Cited by 17 publications

(13 citation statements)

References 87 publications

(91 reference statements)

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“…Likewise, the docking of TcCYSPR04 and TcCYS4 has showed electrostatic complementarities of the surface contact ( Fig 8 ). The same was observed for papain and cystatin S from saliva [ 9 ].…”

Section: Discussionsupporting

confidence: 73%

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TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

et al. 2015

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The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches’ broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.

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Section: Discussionsupporting

confidence: 73%

“…Therefore, the interaction of proteases with their substrates and inhibitors can be seen as a molecular battlefield [ 7 ]. The structure of the complex between exogenous papain and cystatin is well analyzed [ 8 , 9 ], but little is known about the interaction between PLCP and cystatin in the same organism.…”

Section: Introductionmentioning

confidence: 99%

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

et al. 2015

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“…A 500 ps NVT equilibration was performed at temperature of 300 K with position restraints applied to protein and ligand in order to relieve any bad contacts at the residues solvent interface [50]. Then a 1000 ps NPT simulation was conducted, and pressure was coupled to 1.0 atm using the Parrinello−Rahman method.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Anti-Hypertensive Oligopeptides from Adlay Based on In Silico Proteolysis and Virtual Screening

Qiao

Chen

et al. 2016

IJMS

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Adlay (Coix larchryma-jobi L.) was the commonly used Traditional Chinese Medicine (TCM) with high content of seed storage protein. The hydrolyzed bioactive oligopeptides of adlay have been proven to be anti-hypertensive effective components. However, the structures and anti-hypertensive mechanism of bioactive oligopeptides from adlay were not clear. To discover the definite anti-hypertensive oligopeptides from adlay, in silico proteolysis and virtual screening were implemented to obtain potential oligopeptides, which were further identified by biochemistry assay and molecular dynamics simulation. In this paper, ten sequences of adlay prolamins were collected and in silico hydrolyzed to construct the oligopeptide library with 134 oligopeptides. This library was reverse screened by anti-hypertensive pharmacophore database, which was constructed by our research team and contained ten anti-hypertensive targets. Angiotensin-I converting enzyme (ACE) was identified as the main potential target for the anti-hypertensive activity of adlay oligopeptides. Three crystal structures of ACE were utilized for docking studies and 19 oligopeptides were finally identified with potential ACE inhibitory activity. According to mapping features and evaluation indexes of pharmacophore and docking, three oligopeptides were selected for biochemistry assay. An oligopeptide sequence, NPATY (IC50 = 61.88 ± 2.77 µM), was identified as the ACE inhibitor by reverse-phase high performance liquid chromatography (RP-HPLC) assay. Molecular dynamics simulation of NPATY was further utilized to analyze interactive bonds and key residues. ALA354 was identified as a key residue of ACE inhibitors. Hydrophobic effect of VAL518 and electrostatic effects of HIS383, HIS387, HIS513 and Zn2+ were also regarded as playing a key role in inhibiting ACE activities. This study provides a research strategy to explore the pharmacological mechanism of Traditional Chinese Medicine (TCM) proteins based on in silico proteolysis and virtual screening, which could be beneficial to reveal the pharmacological action of TCM proteins and provide new lead compounds for peptides-based drug design.

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“…On the other hand, the acidic pH may also affect the structure of papain. In this sense, the area of contact between the cystatin and the target proteases, in the models of docking, involves hydrophobic and ionic interactions that may be affected by variations in pH [ 36 ]. In addition, many proteins do not support great variations in pH, with denaturation occurring at extreme pH ranges; however, T cCYS4 supported a wide range of variation of pH without losing its inhibitory power completely, proving to be more sensitive to extremely acidic pHs.…”

Section: Discussionmentioning

confidence: 99%

The Activity of TcCYS4 Modified by Variations in pH and Temperature Can Affect Symptoms of Witches’ Broom Disease of Cocoa, Caused by the Fungus Moniliophthora perniciosa

et al. 2015

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The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches’ broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches’ broom disease in cocoa.

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Modelling family 2 cystatins and their interaction with papain

Cited by 17 publications

References 87 publications

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

Discovery of Anti-Hypertensive Oligopeptides from Adlay Based on In Silico Proteolysis and Virtual Screening

The Activity of TcCYS4 Modified by Variations in pH and Temperature Can Affect Symptoms of Witches’ Broom Disease of Cocoa, Caused by the Fungus Moniliophthora perniciosa

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