Satomi Onoue

Mishandling of the Therapeutic Peptide Glucagon Generates Cytotoxic Amyloidogenic Fibrils

AbstractPurpose. Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application. Methods. Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concen- trations ranging from 1 mg/ml to 80 mg/ml and then aged at 37°C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with β-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly. Results. By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a β-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml. Conclusions. These results indicated that glucagon was self-associated by its β-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as β-amyloid (Aβ)1-42 and prion protein fragment (PrP)106-126 including conformational change to a β-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

PACAP protects neuronal PC12 cells from the cytotoxicity of human prion protein fragment 106–126

Misfolding of the prion protein yields amyloidogenic isoforms, and it shows exacerbating neuronal damage in neurodegenerative disorders including prion diseases. Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) potently stimulate neuritogenesis and survival of neuronal cells in the central nervous system. Here, we tested these neuropeptides on neurotoxicity in PC12 cells induced by the prion protein fragment 106–126 [PrP (106–126)]. Concomitant application of neuropeptide with PrP(106–126) (5×10−5 M) inhibited the delayed death of neuron‐like PC12 cells. In particular, PACAP27 inhibited the neurotoxicity of PrP(106–126) at low concentrations (>10−15 M), characterized by the deactivation of PrP(106–126)‐stimulated caspase‐3. The neuroprotective effect of PACAP27 was antagonized by the selective PKA inhibitor, H89, or the MAP kinase inhibitor, U0126. These results suggest that PACAP27 attenuates PrP(106–126)‐induced delayed neurotoxicity in PC12 cells by activating both PKA and MAP kinases mediated by PAC1 receptor.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide attenuate glutamate-induced nNOS activation and cytotoxicity

Both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in the central and peripheral nervous systems. Attention has been focused on these neuropeptides because among their numerous biological activities, they have been confirmed to show neuroprotective effects against ischemia and glutamate-induced cytotoxicity. It is well established that glutamate has excitatory effects on neuronal cells, and that excessive glutamate shows potent neurotoxicity, especially in neuronal nitric oxide synthase-containing neurons. Glutamate stimulates the production of nitric oxide (NO) in neurons, and the NO generated is tightly associated with the delayed death of neurons. We examined the effects of these neuropeptides on the glutamate-induced neural actions using PC12 cells, and we confirmed the important activities of PACAP/VIP on the production of NO as well as the delayed cell death stimulated by glutamate.

The neuromodulatory effects of VIP/PACAP on PC-12 cells are associated with their N-terminal structures.

ONOUE, S., WAKI, Y., NAGANO, Y., SATOH, S., KASHIMOTO, K. Neuromodulatory Effects of VIP/PACAP on PC-12 Cells Are Associated with Their N-terminal Structures. PEPTIDES xx(xx) 000-000, 200x.- The current study explored whether the differences in biological activities in PC-12 cells between vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are attributable to the sequence difference in their N-terminal portions and are correlated with the solution structures of the peptides. In the neurite outgrowth assay, N-terminal modification of VIP to PACAP-like sequences altered its effect, the activity was confirmed even at a low concentration (10(-10) M). On the contrary, N-terminal modification of PACAP 27 to VIP-like sequences reduced its activity. These relationships were also confirmed for the inhibitory effects of the peptide analogues on PC-12 cells growth at 10(-7) M. The present results combined with our previously reported data, including binding assay, support that the N-termini of VIP/PACAP plays an important role in their activities.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactivity in the brain of a teleost, Uranoscopus japonicus: immunohistochemical relationship between PACAP and adenohypophysial hormones

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) could play a role in stimulating pituitary hormone release in fish brain. In this study, we used immunochemical techniques to examine the histological and quantitative distribution of PACAP in the central nervous system (CNS) of a teleost, the stargazer, Uranoscopus japonicus. In addition, high performance liquid chromatographic (HPLC) analysis was performed to characterize the form of PACAP present, while the relationship between PACAP and adenohypophysial hormones was also determined immunohistochemically. PACAP-like immunoreactive (LI) neuronal cell bodies and fibers were found not only in the hypothalamo-pituitary region but also in the midbrain and hindbrain regions. PACAP-LI fibers were identified in the neurohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. The concentration of immunoreactive PACAP in whole brain tissue was approximately 300 pmol/g wet weight. The average concentrations of immunoreactive PACAP in regions of the telencephalon, diencephalon, tectum, cerebellum, and rhombencephalon were 217.53, 510.26, 83.30, 148.64, and 364.62 pmol/g, respectively. In reverse-phase HPLC experiments, the predominant form of immunoreactive PACAP eluted closely with synthetic stargazer PACAP38, while PACAP27-like immunoreactivity was negligible. These results suggest that PACAP38 is the predominant PACAP form in the stargazer CNS, and that PACAP acts not only as a hypophysiotropic factor for adenohypophysial hormone release but also as a neurotransmitter and neuromodulator in the CNS.

Pituitary adenylate cyclase activating polypeptide regulates the basal production of nitric oxide in PC12 cells.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon family, modulate neurotransmission via stimulation of protein kinases including cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the central and peripheral nervous systems. They are reported to co-exist with nitric oxide synthases (NOSs) and other neuropeptides within the nervous system and peripheral tissues. In the present study, we investigated the neuronal role of these peptides in NO production in PC12 cells. We showed that PACAP decreased NO production in a dose-dependent manner, and the activators of protein kinase A and C also inhibited the NO production in PC12 cells. RT-PCR experiments demonstrated that PC12 cells constitutively express the mRNAs for neuronal NOS and the PACAP-specific (PAC1) receptor, and we concluded that PACAP plays an important role in the regulation of nNOS activity through PAC1 receptor in PC12 cells.

Long-acting analogue of vasoactive intestinal peptide, [R15, 20, 21, L17]-VIP-GRR (IK312532), protects rat alveolar L2 cells from the cytotoxicity of cigarette smoke

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in numerous biological responses. We previously reported that the replacement of Lys by Arg, and Met by Leu in VIP (IK312532; [Arg15, 20, 21, Leu17]-VIP) resulted in a significant improvement in metabolic stability and biological activity. In the present study, we investigated the effect of VIP and its related peptides including long-acting VIP derivative (IK312532) and PACAP27 on the cytotoxicity of cigarette smoke extract (CSE), a causative factor of chronic obstructive pulmonary disease (COPD), in rat alveolar L2 cells. RT-PCR displayed the dominant expression of mRNA for the VIP-specific VPAC2 receptor in L2 cells, and VIP and the related peptides showed the specific binding activity and potent stimulation of adenylate cyclase. CSE at a concentration of 0.1% or higher induced significant apoptotic death of L2 cells. Interestingly, the addition of neuropeptides at a concentration of 10(-11) M or higher in L2 cells with CSE (0.25%) resulted in significant attenuation of cell death with the deactivation of CSE-evoked caspase-3 activity. IK312532 was much stable against the enzymatic digestion compared to VIP, and the protective effect of IK312532 was 1.6-fold higher than that of VIP. Taken together with our previous report showing that IK312532 has long-acting relaxant activity in the lung, IK312532 may be a potential candidate for drug treatment of asthma and COPD.

Human antithrombin III-derived heparin-binding peptide, a novel heparin antagonist

In the blood coagulation cascade, human antithrombin III (hAT III) acts as an inhibitor of serine proteases such as thrombin and factor Xa, and this anticoagulatory glycoprotein requires the binding of heparin for its activation. In this study, we synthesized the polypeptides corresponding to the proposed heparin-binding sites including the (41-49), (286-301) and (123-139) regions of hAT III, and examined their interactions with heparin by means of physicochemical and biochemical methods. All the synthetic peptides had a high affinity toward heparin, evidenced by the fact that they were eluted from a heparin-agarose column at the high salt concentration range of 520-700 mM. In addition, hAT III (123-139) attenuated the effect of heparin on the activation of hAT III, whereas other HBPs did not, suggesting that only hAT III (123-139) could interact with the active site of heparin. On the basis of these results, we prepared novel hAT III (123-139)-related derivatives as potent heparin antagonist candidates, and examined the influence of several modifications on their activity in vitro. The results provided new findings about the structure-activity relationship of hAT III (123-139), and led us to the successful development of a potent antagonist for heparin.

Novel approach for preparation of heparins specific to factor Xa using affinity chromatography coupled with synthetic antithrombin III-related peptides

In the blood coagulation cascade, heparin activates human plasma antithrombin III (hAT III), resulting in the inhibition of factor Xa. This polysaccharide also exhibits hemorrhagic tendency mediated by the inhibition of thrombin in heparinotherapy. Therefore, attention has focused on the development of low molecular weight heparins (LMW-heparins) that inhibit factor Xa but not thrombin. In this investigation, we examined the biochemical and physicochemical properties of hAT III-derived heparin-binding peptides (HBPs). Of all the tested HBPs, hAT III (123-139) exhibited the highest affinity with heparin and showed an inhibitory effect on the heparin-induced enhancement of hAT III activity toward factor Xa, indicating that hAT III (123-139) specifically interacts with the active region in heparin. We prepared a synthetic hAT III (123-139)-coupled affinity chromatography system, and demonstrated that this novel affinity chromatography is useful for fractionation of highly active moieties in LMW-heparins.

Imorin: a sexual attractiveness pheromone in female red-bellied newts ( Cynops pyrrhogaster )

The male red-bellied newt (Cynops pyrrhogaster) approaches the female’s cloaca prior to performing any courtship behaviour, as if he is using some released substance to gauge whether she is sexually receptive. Therefore, we investigated whether such a female sexual attractiveness pheromone exists. We found that a tripeptide with amino acid sequence Ala-Glu-Phe is secreted by the ciliary cells in the epithelium of the proximal portion of the oviduct of sexually developed newts and confirmed that this is the major active substance in water in which sexually developed female newts have been kept. This substance only attracted sexually developed male newts and acted by stimulating the vomeronasal epithelial cells. This is the first female sexual attractiveness peptide pheromone to be identified in a vertebrate.