• Journal of Plant Biotechnology
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• 제37권3호
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• pp.327-336
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• 2010

Cysteine proteases have been known as a critical factor in plant defense mechanisms in pineapple, papaya, or wild fig. Papain or ficin is one kind of cysteine proteases that shows toxic effects to herbivorous insects and pathogenic bacteria. However, resistance to bacterial soft rot of plants genetically engineered with cysteine protease has been little examined thus far. We cloned a cysteine protease cDNA from Ananas comosus and introduced the gene into Chinese cabbage (Brassica rapa) under the control of the cauliflower mosaic virus 35S promoter. The transgene was stably integrated and actively transcribed in transgenic plants. In comparisons with wild-type plants, the $T_2$ and $T_3$ transgenic plants exhibited a significant increase in endo-protease activity in leaves and enhanced resistance to bacterial soft rot. A cDNA microarray analysis revealed that several genes were more abundantly transcribed in the transgenic than in the wild type. These genes encode a glyoxal oxidase, PR-1 protein, PDF1, protein kinase, LTP protein, UBA protein and protease inhibitor. These results suggest an important role for cysteine protease as a signaling regulator in biotic stress signaling pathways, leading to the build-up of defense mechanism to pathogenic bacteria in plants.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.69-73
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• 1999

In plants as well as in other multicellular organisms, programmed cell death plays essential roles in the abortion or formation of specific cells and tissues during development to organize the plant [11, 15, 18]. A typical example of developmentally programmed cell death in plants is the death during differentiation of tracheary elements which are components of vessels and tracheids, a water-conducting system. The programming of cell death during tracheary element differentiation has been revealed to be unique to plant cells by using the in vitro Zinnia mesophyll cell culture system. In particular, new biosynthesis of autolysis-related enzymes such as cysteine proteases and nucleases, their accumulation of the vacuole and the programmed collapse of the vacuole are essential to the death of tracheary elements and differ greatly from the process of the apoptotic cell death in animals.

• BMB Reports
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• 제35권2호
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• pp.143-154
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• 2002

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the $\alpha+\beta$ class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0-2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly $\beta$-sheet conformation and shows a strong binding to 8-anilino-1-napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.

• International Journal of Industrial Entomology and Biomaterials
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• 제9권2호
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• pp.217-223
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• 2004

A cathepsin L-like cysteine protease, v-cath, encoded by the baculovirus has been shown to playa role in host liquefaction. We have identified a v-cath gene in the silkworm virus, Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. The 969 bp v-cath has an open reading frame of 323 amino acids. A putative cleavage site and catalytic sites were conserved in BmNPV-K1 v-cath. The predicted three-dimensional structure of BmNPV-K1 v-cath revealed that the overall fold of BmNPV-K1 v-cath is similar to that of other proteases of the papain family. The deduced amino acid sequence of BmNPV-K1 v-cath showed 98% and 97% protein sequence identity to BmNPV T3 strain and to Autographa californica nuclear polyhedrosis virus, respectively. The BmNPV-K1 v-cath differed at 4 amino acid positions from BmNPV T3. The v-cath gene in BmNPV-K1 genome is located on the EcoRV 6 kb and XhoI 9 kb fragments. Northern hybridization analysis of BmNPV K1 v-cath gene revealed that it is expressed late in infection.

• BMB Reports
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• 제35권2호
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• pp.155-164
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• 2002

The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly $\alpha$-helical to $\beta$-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the $\alpha$helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.