Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism.

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.

The propeptide sequence assists the correct folding required for the enzymatic activity of cocoonase.

Cocoonase, a protein that is produced by the silkworm (Bombyx mori), is thought to specifically digest the sericin protein of the cocoon and has a high homology with trypsin. Similar to trypsin, cocoonase is folded as an inactive precursor protein which is activated by releasing the propeptide moiety. However, the mechanism responsible for the activation of its catalytic structure has not yet been determined in detail. Therefore, to investigate the activation and folding mechanism of cocoonase, recombinant cocoonase (CCN) and prococoonase (proCCN) were over-expressed in E. coli cells. Both recombinant proteins (proCCN and CCN) were expressed as inclusion bodies in E. coli cells and their folding was examined under several sets of conditions. After the refolding reactions, both of the recombinant proteins were present as the oxidized soluble forms. The proCCN protein was then auto-processed to release the propeptide region for activation. Interestingly, the CCN (CCN∗) derived from the refolded proCCN showed a much stronger protease activity than the refolded CCN from the reduced CCN in a protease assay using Bz-Arg-OEt as a substrate. In addition, the secondary structure of the refolded CCN protein was similar to that of the CCN∗ protein, as evidenced by CD measurements. These results suggest that the CCN protein becomes trapped in a molten globule-like state without the assistance of the propeptide region during the folding process. We therefore conclude that the propeptide region of CCN kinetically accelerates the folding of CCN to adopt the correct conformation of cocoonase at the final step of the folding pathway.

Repeated treatment with cannabidiol but not Delta9-tetrahydrocannabinol has a neuroprotective effect without the development of tolerance.

Both Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and cannabidiol are known to have a neuroprotective effect against cerebral ischemia. We examined whether repeated treatment with both drugs led to tolerance of their neuroprotective effects in mice subjected to 4h-middle cerebral artery (MCA) occlusion. The neuroprotective effect of Delta(9)-THC but not cannabidiol was inhibited by SR141716, cannabinoid CB(1) receptor antagonist. Fourteen-day repeated treatment with Delta(9)-THC, but not cannabidiol, led to tolerance of the neuroprotective and hypothermic effects. In addition, repeated treatment with Delta(9)-THC reversed the increase in cerebral blood flow (CBF), while cannabidiol did not reverse that effect. Repeated treatment with Delta(9)-THC caused CB(1) receptor desensitization and down-regulation in MCA occluded mice. On the contrary, cannabidiol did not influence these effects. Moreover, the neuroprotective effect and an increase in CBF induced by repeated treatment with cannabidiol were in part inhibited by WAY100135, serotonin 5-HT(1A) receptor antagonist. Cannabidiol exhibited stronger antioxidative power than Delta(9)-THC in an in vitro study using the 1,1-diphenyl-2-picryhydrazyl (DPPH) radical. Thus, cannabidiol is a potent antioxidant agent without developing tolerance to its neuroprotective effect, acting through a CB(1) receptor-independent mechanism. It is to be hoped that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.

Delta9-tetrahydrocannabinol (Delta9-THC) prevents cerebral infarction via hypothalamic-independent hypothermia.

Delta(9)-tetrahydrocannabinol (Delta(9)-THC), a primary psychoactive constituent of cannabis, has been reported to act as a neuroprotectant via the cannabinoid CB(1) receptor. In this study, Delta(9)-THC significantly decreased the infarct volume in a 4 h mouse middle cerebral artery occlusion mouse model. The neuroprotective effect of Delta(9)-THC was completely abolished by SR141716, cannabinoid CB(1) receptor antagonist, and by warming the animals to 31 degrees C. Delta(9)-THC significantly decreased the rectal temperature, and the hypothermic effect was also inhibited by SR141716 and by warming to 31 degrees C. At 24 h after cerebral ischemia, Delta(9)-THC significantly increased the expression level of CB(1) receptor in both the striatum and cortex, but not in the hypothalamus. Warming to 31 degrees C during 4 h cerebral ischemia did not increase the expression of CB(1) receptor at the striatum and cortex in MCA-occluded mice. These results show that the neuroprotective effect of Delta(9)-THC is mediated by a temperature-dependent mechanism via the CB(1) receptor. In addition, warming to 31 degrees C might attenuate both the neuroprotective and hypothermic effects of Delta(9)-THC through inhibiting the increase in CB(1) receptor in both the striatum and cortex but not in the hypothalamus, which may suggest a new thermoregulation mechanism of Delta(9)-THC.

High-cholesterol feeding aggravates cerebral infarction via decreasing the CB1 receptor.

We examined how feeding conditions affect the CB1 receptor and cerebral infarction caused by cerebral ischemia. Mice were divided into the following three groups: normal diet (ND), caloric restriction (CR) and high-cholesterol-enriched diet (HCD), and were kept for 6 weeks. After 6 weeks, we measured both serum and brain cholesterol and the expression level of cannabinoid CB1 receptor within the brain in intact mice. In addition, middle cerebral artery (MCA) was occluded for 2 h following reperfusion. Serum cholesterol significantly increased in the HCD group in comparison with both the ND and CR groups. However, brain cholesterol decreased in the HCD group. Then, the expression level of CB1 receptor significantly decreased in the HCD group, while that of the CR group clearly increased in comparison with the ND group in intact mice. In MCA-occluded mice, The HCD group produced the most severe cerebral infarction, while cerebral infarction was significantly decreased in the CR group. These results suggest that CR prevents infarction by increasing CB1 receptor expression, while high-cholesterol feeding aggravates cerebral infarction both by hypercholesterolemia in serum and by decreasing CB1 receptor expression modulated by hypocholesterolemia within the brain.

Elevated anxiety-like and depressive behavior in Desert hedgehog knockout male mice.

To investigate the functional role of Desert hedgehog (Dhh) gene in the nervous system, we examined motor, sensory, learning and memory functions as well as mood in Dhh knockout (KO) mice. Dhh KO male mice exhibited prolonged immobility time compared with wild-type male mice in the forced swimming test, and showed enhanced inhibition in the Vogel's conflict model. These findings suggest that Dhh KO male mice exhibited enhanced anxiety and depressive behavior compared with wild-type male mice. In contrast, Dhh KO female mice did not show any significant difference compared to wild-type female mice. These behavioral abnormalities of Dhh KO male mice may be due to lower testosterone levels with abnormal development of the testes caused by Dhh-null mutation.

Antipsychotics improve Delta9-tetrahydrocannabinol-induced impairment of the prepulse inhibition of the startle reflex in mice.

Recently, cannabinoid receptor agonists have been reported to impair prepulse inhibition (PPI) of the startle reflex. In the current study, we examined the effect of Delta9-tetrahydrocannabinol (THC), the principal psychoactive component of cannabis, on the PPI, and found that THC (10 mg/kg, i.p.) impaired the PPI concomitant with a decrease in the startle response. Antipsychotics such as haloperidol (0.3 mg/kg, i.p.) and risperidone (0.1 mg/kg, i.p.), which are potent dopamine D2 receptor antagonists, and SR141716 (10 mg/kg, i.p.), a CB1 cannabinoid receptor antagonist, reversed these THC-induced PPI deficits. Moreover, THC (10 mg/kg) increased dopamine (DA) release in the nucleus accumbens but not medial prefrontal cortex over a 50-100-min period (time of PPI test) after treatment, and SR141716 (10 mg/kg) reversed this increase in DA release induced by THC. These results suggest that dopaminergic hyperfunction in the nucleus accumbens may be involved in THC-induced PPI deficits.