The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors

Gruden, Kristina; Štrukelj, Borut; Popović, Tatjana; Lenarčič, Brigita; Bevec, Tadeja; Brzin, Jože; Kregar, I.; Herzog-Velikonja, Jana; Stiekema, Willem J.; Bosch, Dirk; Jongsma, Maarten A.

doi:10.1016/s0965-1748(98)00051-4

Cited by 70 publications

(64 citation statements)

References 44 publications

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“…Considering that tomato CDI does not need complex post-translational modifications to show activity (Brunelle et al, 1999), high-level expression of this PI using such approaches could eventually give more convincing results. Alternatively, inhibitory strategies based on combinations of two or more inhibitors (e.g., Oppert et al, 1993), or on broad-spectrum inhibitors (e.g., Urwin et al, 1998;Gruden et al, 1998) might be necessary, in the short term, to effectively alter protein digestion of Colorado potato beetle in vivo. Inhibition of most cysteine proteinases (Wolfson and Murdock, 1987;Bolter and LatoszekGreen, 1997) or of both cysteine and aspartate proteinase activity (e.g., Gruden et al, 1998) were previously shown to significantly alter growth and development of Colorado potato beetle, presumably by minimizing the number of nontarget (insensitive) proteinases used by the insect to elude the negative effects of PIs.…”

Section: March 2004mentioning

confidence: 99%

“…One effective way to achieve this goal is to identify broad-spectrum inhibitors active against an important fraction of the target pest's protease complement (Gruden et al, 1998), to expand the inhibitory spectrum of inhibitors by grafting complementary inhibitory domains or fusing companion inhibitors (Urwin et al, 1998), or to use mixtures of PIs with complementary inhibitory affinities (Oppert et al, 1993). An alternative way, explored in this study, is to use inhibitors active against those proteinases specifically identified as key enzymes in the initiation of protein digestion (Brunelle et al, 1999;Michaud and Nguyen-Quoc, 2000).…”

Section: Introductionmentioning

confidence: 99%

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Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato

Cloutier

Michaud

2004

Arch Insect Biochem Physiol

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We reported earlier the importance of digestive cathepsin D-like activity for initiating dietary protein hydrolysis in Colorado potato beetle, Leptinotarsa decemlineata Say [Brunelle et al. (1999) Arch. Insect Biochem. Physiol. 42:88-98]. We assessed here whether transgenic lines of potato (Solanum tuberosum L.) expressing a cathepsin D inhibitor (CDI) from tomato would show resistance to the beetle, or if the insect would compensate for the loss of cathepsin D activity after ingesting the recombinant inhibitor. Transgenic potato lines expressing tomato CDI were developed by Agrobacterium tumefaciens genetic transformation, and selected based on their relative amount of CDI. After confirming the absence of detectable visible effects of CDI on the plant's phenotype, diet assays with control and transgenic lines were carried out to assess the impact of the inhibitor on growth and development of the insect. Leaf consumption, relative growth rate, molting incidence, and digestive protease activity were monitored at 12-h intervals over 132 h for 3rd-instar larvae provided with transgenic potato foliage. Leaf consumption and relative growth rate were slightly reduced during the first 12 h for larvae fed CDI, but no significant differences were observed thereafter. In contrast, time for molting to the 4th larval stage was significantly longer for larvae fed modified plants, with developmental delays of approximately 10 h (0.5 day) compared to control larvae. Recombinant CDI also had an impact on the insect's digestive physiology, readily inducing overproduction of digestive proteases (rubiscases), followed by a gradual decrease of total and pepstatin-sensitive activity. Overall, these observations show the ability of Colorado potato beetle to compensate for the loss of cathepsin D activity by modulating its digestive protease complement in response to aspartate-type inhibitors in the diet. From a practical viewpoint, these data stress the importance of devising improved strategies for the effective inhibition of insect digestive proteinases in vivo, based on the use of hybrid inhibitors active against different protease classes.

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Section: March 2004mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato

Cloutier

Michaud

2004

Arch Insect Biochem Physiol

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“…Further studies are needed to establish the precise physiological functions of this new inhibitor in mushrooms. Finally, the discovery of clitocypin should broaden the spectrum of specific cysteine proteinase inhibitors available for potential use in human and veterinary medicine and in agricultural crop protection to fight the remarkable adaptation of insects to plant endogenous inhibitors (51).…”

Section: Table Imentioning

confidence: 99%

Clitocypin, a New Type of Cysteine Proteinase Inhibitor from Fruit Bodies of Mushroom Clitocybe nebularis

Brzin¹,

Rogelj²,

Popović³

et al. 2000

Journal of Biological Chemistry

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A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 M guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. Cysteine proteinases are involved in a diverse array of functions involving specific processing or more general degradation of proteins in a wide variety of organisms, including viruses, fungi, plants, and animals. Their activity is regulated by limited proteolysis of inactive precursors (1, 2), by pH and redox potential of the surroundings, and tight binding with proteinaceous inhibitors (3).Five structurally different groups of protein cysteine proteinase inhibitors have been reported: cystatins (4), thyroglobulin type-1 domain inhibitors or thyropins (5), soybean trypsin inhibitor-like inhibitors of cysteine proteinases from potato (6), pineapple inhibitors of cysteine proteinases (7,8), and very recently, inhibitors of cysteine proteinases homologous to propeptide regions of cysteine proteinases (9). So far, only the mechanism of interaction of the cystatin superfamily of inhibitors has been elucidated (10, 11), followed recently by that of thyropins (12), but overall there is very little information available on all other cysteine proteinase inhibitors concerning specificity, kinetics, and mode of binding.Since cysteine proteinases in mammals have been implicated also in many pathological events, such as tumor invasion and metastasizing cancer (13), bone resorption (14), periodontitis (15), and rheumatoid arthritis (16), there is a need for new specific, efficient, and accessible inhibitors of the enzymes responsible for diagnosis and treatment of these conditions. Fungi (Mycophyta) have been used for religious, medical, and other purposes since ancient times. To our knowledge, no protein cysteine proteinase inhibitors have been characterized in higher fungi (Basydiomyceta), popularly called mushrooms. We report here the identification, some properties, and cloning of a new proteinaceous cysteine proteinase inhibitor from Clitocybe nebularis fruit bodies, which we have called clitocypin, a member of what is very likely a new structural superfamily of cysteine proteinase inhibitors. EXPERIMENTAL PROCEDURESFungal Material-Edible mushrooms, C. nebularis, were collected in their natural habitat in a forest in November and frozen at Ϫ20 or Ϫ70°C until use. A specimen is deposited at the Department of Pharmaceutical Biology, Faculty of Pharmacy, Ljubljana.Chemicals and Enzymes-Iodoacetate and benzoyl-A...

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“…It was our aim to reinforce the defense of crops to pest insects such as S. exigua, by complementing the plant's inhibitor arsenal. We looked for complementary proteinase inhibitors by screening collections of proteinase inhibitors from a wide range of sources [20], or by altering the specificity of plants own inhibitors, by phage display [21]. PI-2-like proteins may constitute a convenient protein framework for the introduction of insecticidal inhibitors into plants.…”

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Characterization of potato proteinase inhibitor II reactive site mutants

Beekwilder

Schipper

Bakker

et al. 2000

European Journal of Biochemistry

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Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (K i 0.4 nm) and chymotrypsin (K i 0.9 nm), and is quite robust towards mutations at positions P2 or P1H . In contrast, the first reactive site inhibits only chymotrypsin (K i 2 nm), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.Keywords: mutational analysis; Pichia pastoris; serine proteinase inhibitor; Solanum tuberosum.Protease inhibitors are abundant proteins in the storage organs and seeds of plants [1]. In addition, their synthesis is induced to high levels in response to stress, infection and wounding [2]. Potato expresses many different proteinase inhibitors belonging to a wide range of inhibitor families. Members of the potato proteinase inhibitor II (PI-2) family have been shown to inhibit serine proteases, such as trypsin, chymotrypsin, subtilisin, oryzin and elastase [3,4]. Until now, they have been found only among the Solanaceae. Proteins and messenger RNAs have been identified in potato tubers [5,6], wounded tomato and tobacco leaves [4,7], eggplant fruits [8], green tomatoes [9] and ornamental tobacco flower stigma [10]. There is medical interest in the properties of PI-2. It has been shown to have anticarcinogenic properties, protecting mouse embryo fibroblasts from radiation-induced transformation [11]. Although the mechanism underlying this effect is poorly understood, it is correlated to the capacity of PI-2 to inhibit chymotrypsin-like enzymes [12].Plant protease inhibitors such as PI-2 have been proposed to function as part of the plant defense system [13]. The plant defense role is deduced from observations that proteinase inhibitors in leaves are synthesized in response to wounding [2,14,15] or viral infection [2,16]. In addition, direct evidence for a protective role has been obtained; it was shown that transgenic tobacco plants over-expressing potato PI-2 gained resistance against the tobacco hornworm Manduca sexta [17]. Also, when the PI-2 gene was introduced into rice under its own wound-inducible promoter, plants were protected from the major lepidopteran rice pest, Sesamia inferens [18]. PI-2 probably acts by inhibiting digestive proteases from the insects, thereby restricting the availability of amino acids [13].The anti-nutrient activity of PI-2 has been shown to be overcome by pest insects like Spodoptera exigua [19]. When these larvae are reared on tobacco leaves expressing potato PI-2, the spe...

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The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors

Cited by 70 publications

References 44 publications

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato

Clitocypin, a New Type of Cysteine Proteinase Inhibitor from Fruit Bodies of Mushroom Clitocybe nebularis

Characterization of potato proteinase inhibitor II reactive site mutants

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