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

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.     

The amino acid sequence of a novel inhibitor of cathepsin D from potato

The amino acid sequence of a cathepsin D inhibitor isolated from potato is described. It was determined by analysis of peptides generated by use of the glycine-specific proteinase PPIV. The order of the peptides was established by examination of tryptic peptides derived from the two cyanogen bromide peptides. The inhibitor comprises 187 amino acid residues, and has a calculated Mr of 20,450.     

A humanized antibody inhibitor for cathepsin L

Cathepsin L (CTSL) is a cysteine protease involved in a variety of physiological and pathological processes. Potent inhibitors against CTSL have long been sought for drug development. Due to insufficient specificity and suboptimal pharmacological properties for current CTSL inhibitors, novel agents are still required for selectively blocking CTSL activity. Here we generated a humanized antibody inhibitor of CTSL by genetically fusing the inhibitory propeptide of procathepsin L to the N‐terminus of the light chain of a humanized antibody. The resulting antibody fusion could be stably expressed and displays highly potent inhibition activity and specificity toward CTSL. This work demonstrates a new approach for the rapid generation of antibody inhibitors of CTSL. It can possibly be extended to create inhibitory antibodies targeting other cathepsin proteases, providing novel research and therapeutic tools.     

Interaction of human cathepsin D with the inhibitor pepstatin.

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.     

Inactivation studies of the leucocyte inhibitor of urokinase by cathepsin D

Leucocytes contain an urokinase inhibitor, that can be inactivated by cathepsin D. In this work biochemical and immunological studies on the inactivation of this inhibitor by cathepsin D are presented. Examinations by polyacrylamide gel electrophoresis and SDS electrophoresis indicate that cathepsin D inactivates urokinase inhibitor by hydrolysis of the inhibitor molecule and that the degradation needed for total inactivation is different from that for formation of the precipitin line with antibodies. The conversion of active inhibitor into inactive protein proceeds catalytically.     

A sensitive fluorometric assay for the determination of cathepsin D

A fluorometric assay is described for cathepsin D—determining the tyrosine released by measuring the fluorescence of its nitrosonaphthol coupling product. The advantage of this method is its high sensitivity.     

Aspartic proteinase inhibitors from tomato and potato are more potent against yeast proteinase A than cathepsin D

The interaction of a variety of aspartic proteinases with a recombinant tomato protein produced in Pichia pastoris was investigated. Only human cathepsin D and, even more potently, proteinase A from Saccharomyces cerevisiae were inhibited. The tomato polypeptide has >80% sequence identity to a previously reported potato inhibitor of cathepsin D. Re-evaluation of the potato inhibitor revealed that it too was more potent (>20-fold) towards yeast proteinase A than cathepsin D and so might be renamed the potato inhibitor of proteinase A. The potency towards yeast proteinase A may reflect a similarity between this fungal enzyme and aspartic proteinases produced by fungal pathogens which attack tomato and/or potatoes.     

Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.     

A novel ditertiary tetrasomic in tomato

Cytogenetic studies were carried out on a ditertiary tetrasomic, 2n + 2 (5L·7S), of tomato (Lycopersicon esculentum Mill.). In studies of transmission of the 5L·7S chromosome, male and female rates of 57.1% and 74.5%, respectively, were found—the highest yet reported for any tomato aneuploid. Additionally, two 5L·7S tertiary chromosomes were transmitted through the female gametophyte, the maximum duplication known to be tolerated by this developmental stage. Monotertiary tetrasomics of a novel type, 2n + 5L·5S + 5L·7S, were recovered in the selfed progeny of 2n + 2(5L·7S). The usefulness of the ditertiary tetrasomic for the production of double trisomics and for dosage and experimental evolutionary studies is elaborated.     

A double-headed cathepsin B inhibitor devoid of warhead

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.     

The effects of lactoyl-pepstatin and the pepsin inhibitor peptide on pig cathepsin D.

Peritoneal macrophages from mice, isolated rat liver Kupffer cells and rat testis Leydig cells ingested large numbers of Percoll particles, a gradient medium widely used for separation of cells and subcellular organelles by density-gradient centrifugation. A decrease in the percentage of macrophages adhering to plastic also occurred after exposure of the cells to Percoll, even at 4 degrees C, a temperature at which Percoll was not ingested. The effect of Percoll on macrophage adherence may involve a loose association between the density medium and the cell surface. Other cell-surface-related phenomena may also be affected by prior exposure of cells to Percoll.     

Brefeldin A inhibits the targeting of cathepsin D and cathepsin H to lysosomes in rat hepatocytes

Effect of brefeldin A on the transport of lysosomal acid hydrolases (cathepsins D and H) was investigated in primary cultured rat hepatocytes. Both cathepsins were synthesized as proenzymes and progressively converted to mature enzymes in the control cells. However, BFA strongly inhibited the appearance of the mature enzymes in the cells in a dose dependent manner, suggesting that transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes is blocked by the drug. The inhibitory effect by brefeldin A was reversible. Upon recovery from brefeldin A-intoxication, procathepsin D was effectively targeted into lysosomes, whereas a substantial amount of procathepsin H was found to be missorted, resulting in its secretion into the culture medium.     

A novel cDNA clone for acid invertase in tomato fruit.

The sequence of a novel cDNA clone, Aiv-1, for tomato acid invertase was similar to that of TIV1 (Klann et al., 1992) for the enzyme except for a unique intron-like insertion. It is considered that Aiv-1 is derived from either an alternatively spliced mRNA for an isozyme or a pre-mRNA of TIV1.     

Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (pepstatin)

Intraperitoneal administration of pepstatin (2 mg/day for 5 weeks) to Lewis rats subjected to experimental allergic encephalomyelitis (EAE) (induced by guinea pig spinal cord and pertussis vaccine) suppressed the appearance of clinical signs of disease, and reduced the severity and incidence of CNS lesions normally associated with this disease. Administration of pepstatin for shorter periods to Lewis rats, or BSVS mice, or guinea pigs challenged with myelin basic protein delayed, but did not prevent clinical signs of EAE, but was accompanied in all cases by a less severe histopathology.